bluuebunny/crossref_metadata_2025 huggingface dataset:
The Contribution of the N-Terminal Structure of Polymyxin B Peptides to Antimicrobial and Lipopolysaccharide Binding Activity
Authors: Sakura Naoki , Itoh Tatsuya , Uchida Yoshiki , Ohki Kazuhiro , Okimura Keiko , Chiba Kenzo , Sato Yuki , Sawanishi Hiroyuki
Published: October 2004

To elucidate the N-terminal structure–activity relationships of polymyxin B peptides, seven polymyxin B component peptides, the structures of which having been elucidated, and seven N-terminal fatty acid and/or amino acid deletion analogs were synthesized, and their antimicrobial activities determined. The lipopolysaccharide (LPS) binding activities of synthetic peptides were evaluated using [Dab(Dansyl-Gly)1]-polymyxin B3 (Dab; l-α,γ-diaminobutyric acid) as a fluorescent probe. The results indicated that the fatty acyl moiety was not indispensable for LPS binding, but the C9 fatty acyl groups of polymyxin B peptides contributed to the binding affinity to a slightly greater extent than C8 or C7. The fatty acyl moieties of polymyxin B contributed greatly to the antimicrobial activity, while the distinct N-terminal structures of polymyxin B1–B6, bearing normal-, iso-, or anteiso-fatty acids, or 3-hydroxy-fatty acid with chain lengths between C7 and C9, did not affect bactericidal potency.
The Synthesis and Biological Activity of Pyranonaphthoquione Derivatives from Streptomyces sp. and Their Related Substances
Authors: Shimbashi Akiko , Tsuchiya Ayako , Imoto Masaya , Nishiyama Shigeru
Published: October 2004

The total synthesis of a novel pyranonaphthoquinone (2) having Cdc25A phosphatase inhibitory activity is achieved. The key step in this synthetic pathway is the intramolecular Michael addition for construction of the pyran ring system. The inhibitory activity of several derivatives, readily obtained from synthetic intermediates of 2, is assessed. The resulting data indicates that the pyran ring moiety is not a crucial factor for the activity.
A Novel Mild Deprotection Method for Phosphine–Boranes
Authors: Schröder Marc , Nozaki Kyoko , Hiyama Tamejiro
Published: October 2004

Treatment of phosphine–boranes with molecular sieves 4Å in a mixture of an ethereal solvent and an alcohol provided deprotected free phosphines in quantitative yields. The phosphines can be obtained by a simple filtration/crystallization procedure in most cases. It should be noted that the current method is successfully applied to the deprotection of a phosphite–borane for the first time.
Reaction of Sterically Crowded Selenoketones with Propiolic Acid
Authors: Okuma Kentaro , Maekawa Shinobu , Nito Yoshinori , Shioji Kosei
Published: October 2004

The reaction of di-t-butyl selenoketone with propiolic acid gave 2H,6H-1,3-oxaselenin-6-one in 78% yield, whereas the reaction of di-t-butyl thioketone with propiolic acid recovered the starting thioketone almost quantitatively.
Preparation of a Binuclear Oxo(tropolonato)molybdenum(V) Complex and Its Spectroscopic Properties and Crystal Structure
Authors: Takekuma Shin-ichi , Tomoda Kengo , Sasaki Masato , Minematsu Toshie , Takekuma Hideko
Published: October 2004

To a solution of tropolone (1) in ethanol was added a solution of Mo2O42+ in 2 M HCl. The mixture was stirred at 25 °C for 15 min yielding the title new complex, [Mo2O4(trop)2] (2), with two molecules of ethanol as ligands.
The Reaction of Butatrienolates with Aldehydes for the Syntheses of α-Vinylidene Acylsilanes
Authors: Shibata Takanori , Takami Kyoko , Arai Yoshikazu , Tsuchikama Kyoji , Maekawa Shunsuke
Published: October 2004

Lithium butatrienolates were prepared in situ by the 1,4-elimination from 2-butynyl trimethylsilyl ethers along with a retro-Brook rearrangement. The addition reaction of the enolates with the aldehydes afforded β-hydroxy-α-vinylidene acylsilanes.
The Pertraction of Hydrophobic Organic Solutes across a Hydrophobic Membrane
Authors: Igawa Manabu , Nemoto Shuji
Published: October 2004

The pertraction of hydrophobic organic solutes across a hydrophobic membrane is a continuous separation process, which can be used as a substitute to active carbon. The permeabilities of hydrophobic organic solutes were examined in a two compartment cell partitioned by a hydrophobic membrane. The permeability was dominated by the hydrophobicity of the solute and the dissociation in the receiving phase. The permeability of the hydrophobic solutes increased with increasing pH of the receiving phase solution for the Teflon membrane filter Fluoropore. The preferential permeability of a hydrophobic solute was obtained, and the pertraction of a hydrophobic solute against its concentration gradient was also possible under a concentration gradient of hydroxide ion. Solute permeability changes were observed by coating the surface of Fluoropore to cover its pores with tetrafluoroethene–hexafluoropropene copolymer, FEP, and a highly selective permeability was obtained between phenol and 2-isopropyl-5-methylphenol (thymol) by suppressing the phenol permeability by coating the FEP membrane.
Large-Scale Production of Ba2+–Alginate-Coated Vesicles of Carbon Nanofibers for DNA-Interactive Pollutant Elimination
Authors: Fugetsu Bunshi , Satoh Shuya , Shiba Toshikazu , Mizutani Taeko , Nodasaka Yoshinobu , Yamazaki Keiji , Shimizu Kiyoko , Shindoh Masanobu , Shibata Ken-ichiro , Nishi Norio , Sato Yoshinori , Tohji Kazuyuki , Watari Fumio
Published: October 2004

Ba2+–alginate coated vesicles (Ba2+–ALG) containing highly dispersed carbon nanofibers (CNFs) were successfully produced for the first time using an encapsulation technique. These Ba2+–ALG/CNFs composite vesicles showed high capabilities in trapping DNA-interactive types of chemicals. For example, 10.0 mL of the vesicles took 0.43 μmol of ethidium ions up from contaminated water within 8 min. Biocompatibility experiments performed in vitro and in vivo provided promising results, suggesting potential applications in in-situ environmental remediation. Kilogram quantities of the Ba2+–ALG/CNFs composite vesicles can be produced within a few hours.
New Morphology-Controlled Poly(aniline) Synthesis Using Anionic Porphyrin Aggregate as a Template and Proton-Driven Structural Changes in the Porphyrin Aggregate
Authors: Hatano Tsukasa , Bae Ah-Hyun , Takeuchi Masayuki , Ikeda Atsushi , Shinkai Seiji
Published: October 2004

It was found that 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS), which is known to aggregate in a one-dimensional fashion in water, acts as a template for the electropolymerization of aniline on an ITO electrode. Examinations using cyclic voltammetry (CV) and UV–vis spectroscopy established that J-aggregated TPPS molecules are entrapped in the resultant poly(aniline). Scanning electron microscopy (SEM) observations established that poly(aniline) grows to a nanosized rod-like structure, reflecting the aggregation structure of TPPS. The chemical oxidative polymerization of aniline in the presence of TPPS also gave a poly(aniline) rod-like structure containing the TPPS J-aggregate. It was found that the J-aggregation mode in the poly(aniline)/TPPS composite disappears when the medium pH is shifted from acidic to basic, but is regenerated when the medium pH is shifted from basic to acidic. This interconversion could be visually recognized by changes in the color and the fluorescence intensity.
Investigations of Phase Structure Transformation and VUV Excitation of YPO4:Tb Synthesized by Solution Precipitation Route
Authors: Di Weihua , Chen Jianyong , Wang Xiaojun , Chen Baojiu
Published: October 2004

Tb-doped YPO4 has been synthesized by a mild and simple solution precipitation approach. pH value of synthesis has a significant influence on the formation and crystallinity of YPO4:Tb. Phase transformation occurs in the annealing process. Vacuum ultraviolet (VUV) excitation of Tb-doped YPO4 is investigated and possible luminescence mechanism under VUV excitation is illustrated.
Synthesis and Spectral Property of Novel Phthalocyanines Substituted with Four Azo Group Moieties on Periphery of Phthalocyanine Ring
Authors: Li Ying-Feng , Li Shao-Lu , Jiang Ke Jian , Yang Lian-Ming
Published: October 2004

A novel class of phthalocyanines substituted with azo group moieties on the periphery of phthalocyanine ring was synthesized, and they show obvious, broad uncommon absorption band in the visible region compared to those normal phthalocyanines.
An Efficient Synthesis of 3,4-Dihydropyran-2-one Derivatives by Lewis Base-catalyzed Tandem Michael Addition and Lactonization
Authors: Tozawa Takashi , Fujisawa Hidehiko , Mukaiyama Teruaki
Published: October 2004

A convenient one-pot preparation of 3,4-dihydropyran-2-one derivatives by Michael addition of silyl enolate derived from phenyl ester with α,β-unsaturated ketones in the presence of a Lewis base catalyst such as tetrabutylammonium phenoxide was developed. In this catalytic cycle, 3,4-dihydropyran-2-ones were produced in good to excellent yields via intramolecular cyclization of in situ formed Michael-adducts.
An Effective Catalyst for Syngas-to-Dimethyl Ether Process with Steamed Zeolite HMCM-49 as Dehydration Component
Authors: Xia Jianchao , Mao Dongsen , Xu Ning , Chen Qingling , Zhang Yahong , Tang Yi
Published: October 2004

The HMCM-49 zeolites before and after steam treatment were commingled with methanol synthesis catalyst to perform the single-step syngas-to-dimethyl ether process. The high temperature steam treatment meliorated the acidity of HMCM-49 zeolite, and consequently enhanced the selectivity to dimethyl ether significantly by reducing the formation of by-products like carbon dioxide and hydrocarbons.
Synthesis of Wollastonite Single Crystal Nanowires by a Novel Hydrothermal Route
Authors: Li Xiaoke , Chang Jiang
Published: October 2004

This article firstly reports the successful synthesis of wollastonite single crystal nanowires by hydrothermal route. We obtained xonotlite nanowires on hydrothermal condition at a lower temperature (180 °C) using cationic surfactant as template. The nanowires have high aspect ratios more than 100 with diameters of 50–100 nm. After calcination at 800 °C for 2 h, xonotlite nanowires transformed to wollastonite and the fibrous structure was preserved. They were characterized by powder X-ray diffractometry (XRD), transmission electron microscopy (TEM). The mechanism of wollastonite nanowires formation was also discussed.
Synthesis, Structure, and Magnetic Property of Organic-radical Labeled Carborane
Authors: Iwahori Fumiyasu , Kamibayashi Kengo , Nishikawa Yoshikazu , Yamashita Masahiro , Abe Jiro
Published: October 2004

A carborane derivative containing nitronyl nitroxide group was synthesized and was crystallographically and magnetically characterized. The X-ray structure analysis revealed that the molecules were crystallized in a head-to-tail dimer fashion. The intradimer ferromagnetic interaction (J/kB = +4.26(2) K) was observed.
Formation of Stable Molecular Glasses of Yttrium(III) Acyl-Dl-Alaninate Complexes
Authors: Iida Masayasu , Masuda Rie , Naren Gerile , Bin Yuezhen , Kajiwara Kazuhito
Published: October 2004

Yttrium(III) acylalaninate complexes such as tris(N-hexanoyl-Dl-alaninato)yttrium(III) (=Y(hex-Dl-ala)3) and tris(N-octanoyl-Dl-alaninato)yttrium(III) (=Y(oct-Dl-ala)3) form stable glassy states by an evaporation of solvent from the methanol solution. The trend of formation of the stable glassy state is sensitive to a difference in the acylalaninate ligands.
Polarity of Room-Temperature Ionic Liquid as Examined by EPR Spectroscopy
Authors: Kawai Akio , Hidemori Takehiro , Shibuya Kazuhiko
Published: October 2004

Polarity of several ionic liquids was examined by 14N hyperfine coupling constant of nitroxide radical measured by EPR method. Polarity judged from EPR agrees well with those by solvatochromic dyes, suggesting that EPR is another valuable probe for polarity of ionic liquids.
A Synthetic Peptide Derived from the Non-Structural Protein 3 of Hepatitis C Virus Serves as a Specific Substrate for PKC
Authors: Borowski Peter , Resch Kerstin , Schmitz Herbert , Heiland Max
Published: October 2004

A synthetic peptide corresponding to the amino acid sequence Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500of the hepatitis C virus (HCV) polyprotein was found to be a selective substrate for protein kinase C (PKC). In the presence of Ca2+, TPA and phospholipid, PKC phosphorylates the peptide [termed HCV(1487–1500)] with aKmof 11μM andVmaxof 24 μmol × min−1× mg−1. HCV(1487–1500) acts as a competitive inhibitor of PKC towards other peptide or protein substrates and inhibits the kinase activity with an IC50corresponding to theKmvalues measured for the substrates. N- or C-terminally deleted analogs of HCV(1487–1500) did not show inhibitory effects and were only marginally or not phosphorylatable.

We designed an additional peptide in which the tyrosine residue was replaced by phenylalanine ([Phe1499]HCV(1487–1500)). This peptide was neither phosphorylated by other serine/threonine kinases tested nor by whole cell extracts prepared from PKC-depleted cells. [Phe1499]HCV(1487–1500) was used to monitor the TPA-induced translocation of PKC activity to the particulate fraction in JB6 cells. The use of SDS/PAGE to separate the peptide from ATP and Piallowed to monitor simultaneously PKC autophosphorylation and phosphorylation of the peptide. The data presented here show that [Phe1499]HCV(1487–1500) can serve as a convenient tool for investigations of PKC activity also in the presence of other kinases in tissues or in crude cell extracts.
Activation of Progelatinase B by Membranes of Human Polymorphonuclear Granulocytes
Authors: Kolkenbrock Hansjörg , Zimmermann Jürgen , Burmester Gerd-R. , Ulbrich Norbert
Published: October 2004

Isolated human granulocyte plasma membranes contain progelatinase B. The binding of progelatinase B to the membrane, however, is relatively weak, and a considerable part of progelatinase B can be removed by simply washing the membrane with buffer. This detachment does not depend on the ionic strength of the buffer, indicating that electrostatic forces do not play an important role in the binding of progelatinase B to the membrane. A complete removal of progelatinase B is achieved by chromatography of neutrophil membranes on gelatin-agarose.

The plasma membrane of human granulocytes activates added progelatinase B. This activation is inhibited by soybean trypsin inhibitor and is thus performed by membrane bound serine proteinases. In contrast to other reports that claimed an important role of elastase in activating progelatinase B, we found that this activation is mostly inhibited by chymostatin and not by elastatinal and is thus primarily due to cathepsin G. Proteinase 3 was shown to activate progelatinase B as efficient as neutrophil elastase, i. e. much weaker than cathepsin G. Binding of cathepsin G and elastase to the neutrophil membrane does not change their ability to activate progelatinase B. However, cathepsin G , the most potent activator of the three neutrophil serine proteinases, is only a weak activator, when compared to stromelysin-1. This, as well as only a weak binding of progelatinase B, make it doubtful that activation of membrane-bound progelatinase B by membrane-bound serine proteinases is of significant physiological importance.
Tissue-Specific Regulation of IRS-2/PI 3-Kinase Association in Aged Rats
Authors: Carvalho Carla R.O. , Maeda Lucimara , Brenelli Sigisfredo L. , Saad Mario J.A.
Published: October 2004

We have examined the insulin-stimulated IRS-2 association with PI 3-kinase and the phosphorylation of AKT/PKB, which is functionally located downstream of the PI 3-kinase, in aged (obese) rats. The IRS-2 protein levels were similar in 2 and 20 month-old rats in both tissues, liver and muscle. There were reductions in insulin-induced IRS-2 tyrosine phosphorylation in liver and muscle, accompanied by a decrease in IRS-2/PI 3-kinase association and in AKT/PKB phosphorylation only in muscle tissue of aged rats. This regulation may be important in the altered glucose metabolism observed in aged (obese) rats.
Investigation of the Effect of Freezing on Protease-Catalyzed Peptide Synthesis Using Cryoprotectants and Frozen Organic Solvent
Authors: Haensler Marion , Arnold Klaus
Published: October 2004

In order to investigate the effect of freezing on aqueous protease-catalyzed peptide synthesis systems, the influence of polyethylene glycols as cryoprotecting substances on α-chymotrypsin-catalyzed coupling of a N-protected acyl donor ester and various nucleophilic amino components was studied. Changes in S'-specificity of α-chymotrypsin in frozen aqueous systems were suppressed by polyethylene glycols even at concentrations below 1% (w/v). Furthermore, the influence of freeze-concentration in organic solvents on protease-catalyzed peptide synthesis was investigated for the first time. In frozen tert-butanol, α-chymotrypsin-catalyzed peptide synthesis took advantage from freeze-concentration, but in contrast to frozen aqueous systems, no changes in S'-specificity of the biocatalyst were observed. The results suggest that freeze-concentration is not the only cause of freezing-induced yield improvement in aqueous peptide synthesis systems, but interactions between enzyme and ice structures strongly contribute to the observed effects.
The Twin-Arginine Translocation System: A Novel Means of Transporting Folded Proteins in Chloroplasts and Bacteria
Authors: Robinson C.
Published: October 2004

Protein translocases have been characterised in several membrane systems and the translocation mechanisms have been shown to differ in critical respects. Nevertheless, the majority were believed to transport proteins only in a largely unfolded state, and this widespread characteristic was viewed as a likely evolutionary effort to minimise the diameter of translocation pore required. Within the last few years, however, studies on the chloroplast thylakoid membrane have revealed a novel class of protein translocase which possesses the apparently unique ability to transport fullyfolded proteins across a tightly sealed energytransducing membrane. A related system, (the twinarginine translocation, or Tat system) has now been characterised in the Escherichia coli plasma membrane and considerations of its substrate specificity again point to its involvement in the transport of folded proteins. The emerging data suggest a critical involvement in many membranes for the biogenesis of two types of globular protein: those that are obliged to fold prior to translocation, and those that fold too tightly or rapidly for other types of protein translocase to handle.
Responses to Peroxynitrite in Yeast: Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) as a Sensitive Intracellular Target for Nitration and Enhancement of Chaperone Expression and Ubiquitination
Authors: Buchczyk Darius P. , Briviba Karlis , Hartl F. Ulrich , Sies Helmut
Published: October 2004

Peroxynitrite (ONOO−), a potent oxidizing and nitrating species, has been linked to covalent modifications of biomolecules in a number of pathological conditions. InS. cerevisiae, a model eukaryotic cell system, ONOO−was found to be more potent than hydrogen peroxide in oxidizing thiols, inducing heat shock proteins (Hsp70) and enhancing the ubiquitination of proteins. As identified by microsequence analysis following immunoprecipitation with anti-nitrotyrosine antibodies, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was especially susceptible to nitration by ONOO−in yeast cells. The activity of this enzyme was strongly inhibited upon steady-state exposure of the cells to low doses of ONOO−in yeast and in cultured rat astrocytes. Thus, ONOO−is a potent stressor in yeast capable of inducing oxidative damage and protein nitration, with GAPDH being a preferential target protein that is efficiently inactivated.
Enhancing the T R Transition of Insulin by Helix-Promoting Sequence Modifications at the N-Terminal B-Chain
Authors: Shneine Jewad , Voswinkel Marc , Federwisch Matthias , Wollmer Axel
Published: October 2004

Structurally, the T→R transition of insulin mainly consists of a rearrangement of the N-terminal B-chain (residues B1–B8) from extended to helical in one or both of the trimers of the hexamer. The dependence of the transition on the nature of the ligands inducing it, such as inorganic anions or phenolic compounds, as well as of the metal ions complexing the hexamer, has been the subject of extensive investigations. This study explores the effect of helix-enhancing modifications of the N-terminal B-chain sequence where the transition actually occurs, with special emphasis on N-capping. In total 15 different analogues were prepared by semisynthesis. 80% of the hexamers of the most successful analogues with zinc were found to adopt the T3R3state in the absence of any transforming ligands, as compared to only 4% of wild-type insulin. Transformation with SCN−ions can exceed the T3R3state where it stops in the case of wild-type insulin. Full transformation to the R6state can be achieved by only one-tenth the phenol concentration required for wild-type insulin, i. e. almost at the stoichiometric ratio of 6 phenols per hexamer.
3-Hydroxybenzoate:Coenzyme A Ligase from Cell Cultures of Centaurium erythraea: Isolation and Characterization
Authors: Barillas Wagner , Beerhues Ludger
Published: October 2004

In xanthone biosynthesis, 3-hydroxybenzoate:coenzyme
A ligase (3HBL) supplies the starter substrate for
the formation of an intermediate benzophenone. 3HBL
from cell cultures of the medicinal plant Centaurium
erythraea was purified to apparent homogeneity using
a sevenstepprocedure. The enzyme was an AMPforming CoA ligase with a K = 14.7 for 3-hydroxybenzoic acid, 8.5 for coenzyme A and 229 for ATP. The pH and temperature optima were 7.5 and 35C, respectively. In SDSPAGE, two polypeptides of M 41500 and 40500 were detected. Both proteins were structurally related to each other as shown by tryptic digestion. Their Ntermini were blocked. The difference in their apparent molecular masses could
not be attributed to glycosylation. 3HBL had a native
M of approx. 50000 and is thus active as a monomer.
Permutation of the Active Site Motif of Tryparedoxin 2
Authors: Steinert Peter , Plank-Schumacher Karin , Montemartini Marisa , Hecht Hans-Jürgen , Flohé Leopold
Published: October 2004

Tryparedoxins (TXN) are thioredoxinrelated proteins which, as trypanothione:peroxiredoxin oxidoreductases, constitute the trypanothionedependent antioxidant defense and may also serve as substrates for ribonucleotide reductase in trypanosomatids. The active site motif of TXN2, [40]WCPPCR[45], of Crithidia fasciculata was mutated by sitedirected mutagenesis and eight corresponding muteins were expressed in E. coli as terminally Histagged proteins, purified to homogeneity by nickel chelate chromatography, and characterized in terms of specific activity, specificity and, if possible, kinetics. Exchange of Cys41 and Cys44 by serine yielded inactive products confirming their presumed involvement in catalysis. Exchange of Arg45 by aspartate resulted in loss of activity, suggesting an activation of active site cysteines by the positive charge of Arg45. Substitution of Trp40 by phenylalanine or tyrosine resulted in moderate decrease of specific activity, as did exchange of Pro42 by glycine. Kinetic analysis of these three muteins revealed that primarily the reaction with trypanothione is affected by the mutations. Simulation of thioredoxin or glutaredoxin like active sites in TXN2 (P42G and W40T/P43Y, respectively) did not result in thioredoxin or glutaredoxin like activities. These data underscore that TXNs, although belonging to the thioredoxin superfamily, represent a group of enzymes distinct from thioredoxins and glutaredoxins in terms of specificity, and appear attractive as molecular targets for the design of trypanocidal compounds.
In Vitro Evolution of Ligands for HCV-Specific Serum Antibodies
Authors: Zhu Zong Yu , Minenkova Olga , Bellintani Francesca , Tomassi Amedeo De , Urbanelli Lorena , Felici Franco , Monaci Paolo
Published: October 2004

We developed a strategy to improve the properties of ligands selected from phagedisplayed random peptide libraries. A sitedirected mutagenesis protocol that introduces mutations and extends the size of a target sequence has been set up to generate diversity in a single or in a population of clones. The pool of mutants thus created is screened to identify variants with the desired properties. We refer to this strategy as in vitro evolution of ligands. Here we report the application of this in vitro evolution protocol to the identification of improved ligands for HCVspecific serum antibodies. A single clone or population of clones were processed to generate a secondary library. Screening of these libraries with sera from HCVinfected patients identified peptides with an enhanced and broadened ability to detect HCVspecific serum antibodies.
Stability of Bacteriophage T4 Short Tail Fiber
Authors: Burda Martin R. , Hindennach Ingrid , Miller Stefan
Published: October 2004

Adsorption of Teven bacteriophages to the E. coli
host cell is mediated by long and short tail fibers. Bacteriophage T4 short tail fiber protein p12 was used to
investigate the stability against thermal and chemical
denaturation. Purified p12 is thermostable with a melting
point of 78C. Guanidinium chlorideinduced denaturation
displayed strong hysteresis and an intermediate
between 2 and 3 denaturant. The transitions
occur at 1.5 and 3.2 denaturant as revealed by fluorescence spectroscopy and circular dichroism. The
data suggest an equilibrium unfolding intermediate
with a separate unfolding of the Cterminal knob domain
and the shaft region.
Guanosine Nucleotides Regulate B2 Kinin Receptor Affinity of Agonists But Not of Antagonists: Discussion of a Model Proposing Receptor Precoupling to G Protein
Authors: Faussner Alexander , Roscher Adelbert A.
Published: October 2004

The effect of nucleotides on binding of the B2 kinin (BK) receptor agonist [[3]H]BK and the antagonist [[3]H]NPC17731 to particulate fractions of human foreskin fibroblasts was studied. At 0C, particulate fractions exhibited a single class of binding sites with a K of 2.3n for [[3]H]BK and a K of 3.8n for the antagonist [[3]H]NPC17731. Incubation with radioligands at 37C for 5 min gave a reduction of agonist, as well as antagonist, binding that was between 0 40% depending on the preparation, even in the absence of guanosine nucleotides. As shown by Scatchard analysis, this reduction in specific binding was due to a shift in the affinity of at least a fraction of the receptors. The presence at 37C of the guanine nucleotides GTP, GDP and their poorly hydrolyzable analogs left [[3]H] NPC17731 binding unaffected, but reduced the receptor affinity for [[3]H]BK to a K of about 15 n. The maximal number of receptors, however, was unchanged. This affinity change was strongly dependent on the presence of bivalent cations, in particular Mg[2+]. It was reversed by incubation at 0C. The rank order of the guanosine nucleotides for [[3]H]BK binding reduction was GTP[γS] = Gppp [greater than] GTP = GDP [greater than] GDP[βS]. GMP, ATP, ADP and AMP showed no influence on agonist binding. A model for the interaction of the B2 kinin receptor with G proteins is discussed.
Regulation of PTHrP and PTH/PTHrP Receptor by Extracellular Ca2+Concentration and Hormones in the Breast Cancer Cell Line 8701-BC
Authors: Luparello Claudio , Santamaria Francesca , Schilling Tobias
Published: October 2004

It was previously reported that 8701-BC breast tumour cells express the gene for parathyroid hormonerelated peptide (PTHrP) and PTH/PTHrP receptor (PTHrPR) and release immunoreactive PTHrP (iPTHrP) into the extracellular medium. Since the regulation of PTHrP and PTHrPR by breast cancer cells has been poorly investigated so far, we have chosen the 8701- BC cell line as a model system to investigate whether alterations in the extracellular Ca[2+] concentration ([Ca[2+]]) and treatment with some wellknown differentiation agents for breast cells, such as dimethyl sulfoxide, hydrocortisone, progesterone, prolactin, alltrans retinoic acid and transforming growth factorβ1 might (i) modulate quantitatively the release of iPTHrP, (ii) affect the PTHrP promoter usage and mRNA splicing patterns, and (iii) modify the expression of PTHrPR. The data obtained indicate that 8701-BC cells are potentially able to utilise different start sites and mRNA splicing patterns for PTHrP transcription, and respond to variations of [Ca[2+]] and to the addition of two hormones, hydrocortisone and progesterone, with modifications in the extracellular amount of iPTHrP. Moreover, expression of PTHrPR is also modulated by changes of [Ca[2+]] or treatment with hydrocortisone. This indicates that the 8701-BC cell line is a suitable in vitro model for further studies on the complex molecular regulation of the PTHrP/PTHrPR pair in breast cancer.
Urethanyl-3-Amidinophenylalanine Derivatives as Inhibitors of Factor Xa. X-Ray Crystal Structure of a Trypsin/Inhibitor Complex and Modeling Studies
Authors: Sperl Stefan , Bergner Andreas , Stürzebecher Jörg , Magdolen Viktor , Bode Wolfram , Moroder Luis
Published: October 2004

Hydrophobic urethanyl derivatives of 3-amidinophenylalanine methyl ester were found to be relatively potent and selective factor Xa inhibitors. These compounds consist of the argininemimetic 3-benzamidino group as P1 residue and of hydrophobic residues as potential interaction partners for the S3/S4 aryl binding site of the enzyme. Attempts to possibly identify their binding mode to factor Xa via the Xray crystal structure of a trypsin/inhibitor complex and analogy modeling on the crystal structure of factor Xa failed. However, synthesis of enantiomerically pure (R) and (S)derivatives, combined with modeling experiments, led to an hypothetical nonsubstrate like binding mode, which was fully confirmed by the remarkably inhanced inhibitory potency of derivatives in which the methyl ester was replaced by arylamides for interactions with the S3/S4 enzyme binding subsites. With adamantyloxycarbonyl(R)-3-amidinophenylalaninephenethylamide a nanomolar inhibiton was obtained, thus indicating this new class of factor Xa inhibitors as a highly promising lead structure.
The pH-Dependent Interaction of Cinnamomin with Lipid Membranes Investigated by Fluorescence Methods
Authors: Hu Rong-gui , Tang Shuang , Liu Wang-yi
Published: October 2004

Cinnamomin, a new type II ribosomeinactivating protein (RIP), was found to be able to induce the release of calcein loaded in lecithin small unilamellar vesicles and the fusion or aggregation of the lecithin liposomes. Such induction could be promoted several fold by a pH 5.0 environment, a condition similar to that in endocytic vesicles. Lowering the pH from 7.5 to 5.0 evoked conformational changes of cinnamomin and unmasked its hydrophobic areas, including the exposure of 1-anilino-8-naphthalenesulfonate (1,8-ANS) binding sites of the molecule. Some tryptophan residues with affinity to acrylamide were demonstrated to participate in the lipidprotein interaction. The pH dependent fusogenicity of type II RIP might suggest its in vivo function as a fusogen to exert its cytotoxicity.
Nitric Oxide Detection and Visualization in Biological Systems. Applications of the FNOCT Method
Authors: Meineke Petra , Rauen Ursula , Groot Herbert de , Korth Hans-Gert , Sustmann Reiner
Published: October 2004

Fluorescent Nitric Oxide Cheletropic Traps (FNOCTs)
were applied to specifically trap nitric oxide (NO) with
high sensitivity. The fluorescent oquinoid ?electron
system of the FNOCTs (? = 460 nm, ? = 600 nm) reacts
rapidly with NO to a fluorescent phenanthrene
system (? = 380 nm, ? = 460 nm). The cyclic nitroxides
thus formed react further to nonradical products
which exhibit identical fluorescence properties. Using
the acid form of the trap (FNOCT-4), NO release by
spermine NONOate and by lipopolysaccharide (LPS)
activated alveolar macrophages were studied. A maximum
extracellular release of NO of 37.5 nmol h[-1] (10[6]
cells)[-1] from the macrophages was determined at 11 h
after activation. Furthermore, intracellular NO release
by LPSactivated macrophages and by microvascular
omentum endothelial cells stimulated by the Ca[2+]
ionophore A-23187, respectively, was monitored on
the single cell level by means of fluorescence microscopy.
After loading the cells with the membranepermeating
acetoxymethylester derivative FNOCT-5,which is hydrolyzed to a nonpermeating dicarboxylate
by intracellular hydrolases, NO formation by the
endothelial cells started immediately upon stimulation,
whereas start of NO production by the macrophages
was delayed with a variation between 4 and 8 h
for individual cells. These results demonstrate that the
FNOCTs can be used to monitor NO release from
single cells, as well as from NOdonating compounds,
with high sensitivity and with temporal and spatial
resolution.
Multiple Promoters Direct the Tissue-Specific Expression of Rat Mitochondrial Glycerol-3-Phosphate Dehydrogenase
Authors: Weitzel Joachim M. , Grott Stefan , Radtke Christiane , Kutz Sabine , Seitz Hans J.
Published: October 2004

The mitochondrial FADdependent glycerol-3-phosphate dehydrogenase (mGPDH) is an essential component of the glycerol phosphate shuttle which transfers reduction equivalents from the cytoplasm into the mitochondria. We analyzed the distribution of different exon 1-containing transcripts by RTPCR in various tissues in vivo. Exon 1a was predominantly expressed in brain, brown adipose tissue and pancreas, exon 1b was ubiquitously expressed, and exon 1c was exclusively expressed in testis. In transient transfection assays the ubiquitous promoter B showed a detectable activity, whereas promoters A and C were completely silent. A deletion mutational analysis located the basal promoter B activity to a 316 bp core sequence upstream of the transcription start site.
Characterization of SNP, a Novel Tissue- and Phase-Specific Nuclear Protein Expressed during the Proliferative Phase in the Oviduct of the Lizard Podarcis sicula Raf.
Authors: Quesada Piera , Atorino Luigia , Parente Augusto , Blanco Francesca Del Vecchio , Maro Antimo Di , Ciarcia Gaetano , Farina Benedetta
Published: October 2004

The amino acid sequence of a novel tissueand phasespecific nuclear protein (SNP) has been determined, after purification from the nuclei of the oviduct of the lizard Podarcis sicula Raf. during the reproductive period of the seasonal growth. SNP has a pI of 9.0 and contains 81 amino acid residues with a molecular weight of 9211.88 ± 0.09. It shows a bipartite organization as the first 40 amino acids contain all 8 cysteinyl residues, while the last 41 amino acids contain 16 prolyl residues. Two more components have also been identified and characterized, with the first 79 amino acids matching SNP and missing one or two residues at the Cterminus. They have thus been named [des(Ala[81]) SNP1] and [des(Lys[80]Ala[81]) SNP2], respectively. The molecular weights are 9140.21 ± 0.83 for [des(Ala[81]) SNP1] and 9011 ± 0.09 for [des(Lys[80]Ala[81]) SNP2].
Re-Evaluation of Amino Acid Sequence and Structural Consensus Rules for Cysteine-Nitric Oxide Reactivity
Authors: Ascenzi Paolo , Colasanti Marco , Persichini Tiziana , Muolo Massimo , Polticelli Fabio , Venturini Giorgio , Bordo Domenico , Bolognesi Martino
Published: October 2004

Nitric oxide (NO), produced in different cell types through
the conversion of Larginine into Lcitrulline by the enzyme NO synthase, has been proposed to exert its action in several physiological and pathological events.
The great propensity for nitrosothiol formation and
breakdown represents a mechanism which modulates
the action of macromolecules containing NOreactive
Cys residues at their active centre and/or allosteric
sites. Based on the human haemoglobin (Hb)
structure and accounting for the known acidbase
catalysed Cys?93-nitrosylation and Cys?93NOdenitrosylation processes, the putative amino acid sequence (Lys/Arg/His/Asp/Glu)Cys(Asp/Glu) (sites 1,
0, and + 1, respectively) has been proposed as the minimum consensus motif for CysNO reactivity. Although
not found in human Hb, the presence of a polar amino
acid residue (Gly/Ser/Thr/Cys/Tyr/Asn/Gln) at the 2
position has been observed in some NOreactive protein
sequences (e.g., NMDA receptors). However, the most important component of the tri or tetrapeptide consensus motif has been recognised as the Cys(Asp/Glu) pair [Stamler et al., Neuron (1997) 18,
691 696]. Here, we analyse the threedimensional
structure of several proteins containing NOreactive Cys residues, and show that their nitrosylation and denitrosylation processes may depend on the CysS? atomic structural microenvironment rather than on the tri or tetrapeptide sequence consensus motif.
Expression of Human Tissue Kallikrein in Differentiated HL-60 Cells
Authors: Podlich Dirk , Kemme Michael
Published: October 2004

The kallikreinkinin system is a mediator of inflammation in humans. In order to elucidate the range of expression of human tissue kallikrein and its substrates, high and low molecular weight kininogen, in inflammatory cells in vitro, we examined their biosynthesis in the HL-60 cell line by RTPCR and Southern blot analyses. Prominent expression of tissue kallikrein mRNA occurred in untreated promyelocytic cultures as well as in HL-60 cells that were induced to differentiate toward neutrophilic, monocytic, and macrophagic cells. Under the same inducing conditions, kininogen biosynthesis was undetectable at each differentiation state of HL-60 cultures. These results indicate that the myelomonocytic lineage of human leukocytes is a source of tissue kallikrein, which may be secreted as part of the inflammatory process.
Protein Aggregation and Pathogenesis of Huntingtons Disease: Mechanisms and Correlations
Authors: Wanker Erich E.
Published: October 2004

The formation of insoluble protein aggregates is a hallmark
of Huntington's disease (HD) and related neurodegenerative
disorders, such as dentatorubral pallidoluysian
atrophy (DRPLA), spinal bulbar muscular
atrophy (SBMA) and the spinocerebellar ataxia (SCA)
type 1, 2, 3, 6 and 7. These disorders are caused by an
expanded polyglutamine (polyQ) tract in otherwise unrelated
proteins. They are characterized by late-onset,
selective neuropathology, a pathogenic polyQ threshold
and a relationship between polyQ length and disease
progression. Thus, molecular models of HD and
related glutamine-repeat disorders must account for
these characteristic features. During the last three
years, considerable effort has been invested in the
development of in vitro and in vivo model systems to
study the mechanisms of protein aggregation in glutamine-repeat disorders and its potential effects on disease
progression and neurodegeneration. A selection
of these studies is reviewed here. Furthermore, the
correlation between aggregate formation and development
of HD is discussed.
Molecular Genetic Analysis of Glucocorticoid Signaling Using the Cre/loxP System
Authors: Reichardt Holger M. , Tronche François , Bauer Anton , Schütz Günther
Published: October 2004

Glucocorticoids (GC) are involved in a plethora of
physiological processes that range from the regulation
of the stress response and the control of the immune
system to modulation of behavior. Most GC effects
are mediated by the glucocorticoid receptor (GR)
via activation and repression of gene expression.
Whereas in most cases activation requires DNA binding
of the receptor, repression is usually mediated by
protein-protein interaction with other transcription
factors. To decipher the molecular mode of action of
GR, mice were generated by gene targeting carrying a
point mutation in one of the dimerization domains, thus
abrogating DNA binding by GR. Analysis of these mice
demonstrated that thymocyte apoptosis and stress
erythropoiesis require the DNA binding-dependent
function of GR, whereas lung development and the
anti-inflammatory activity of GR are mediated by protein-protein interaction. Furthermore, to study the role
of GC in the brain, mice were generated specifically
lacking GR function in the nervous system. Using
these mice we demonstrated that GR is essential for
the regulation of the HPA-axis and the stress response,
as well as for the control of emotional behavior. Taken
together, gene targeting using the Cre/loxP system
proved to be highly valuable for the analysis of both
molecular mechanism and tissue-specific functions
of the GR.
Macromolecular Intelligence in Microorganisms
Authors: Bruggeman Frank J. , Heeswijk Wally C. van , Boogerd Fred C. , Westerhoff Hans V.
Published: October 2004

Biochemistry and molecular biology have been focusing on the structural, catalytic, and regulatory properties of individual macromolecules from the perspective of clarifying the mechanisms of metabolism and gene expression. Complete genomes of ‘primitive’ living organisms seem to be substantially larger than necessary for metabolism and gene expression alone. This is in line with the findings of silent phenotypes for supposedly important genes, apparent redundancy of functions, and variegated networks of signal transduction and transcription factors. Here we propose that evolutionary optimization has been much more intensive than to lead to the bare minima necessary for autonomous life. Much more complex organisms prevail. Much of this complexity arises in the nonlinear interactions between cellular macromolecules and in subtle differences between paralogs (isoenzymes). The complexity can only be understood when analyzed quantitatively, for which quantitative experimentation is needed in living systems that are as simple and manipulatable as possible, yet complex in the above sense. We illustrate this for the glutamine synthetase cascade inEscherichia coli. By reviewing recent molecular findings, we show that this cascade is much more complex than necessary for simple regulation of ammonia assimilation. Simulations suggest that the function of this complexity may lie in quasi-intelligent behavior, including conditioning and learning.
Helical Tubes of FtsZ from Methanococcus jannaschii
Authors: Löwe Jan , Amos Linda A.
Published: October 2004

Bacterial cell division depends on the formation of a cytokinetic ring structure, the Z-ring. The bacterial tubulin homologue FtsZ is required for Z-ring formation. FtsZ assembles into various polymeric formsin vitro, indicating a structural role in the septum of bacteria. We have used recombinant FtsZ1 protein fromM. jannaschiito produce helical tubes and sheets with high yield using the GTP analogue GMPCPP [guanylyl-(α,β)-methylene-diphosphate]. The sheets appear identical to the previously reported Ca++-induced sheets of FtsZ fromM. jannaschiithat were shown to consist of ‘thick’-filaments in which two protofilaments run in parallel. Tubes assembled either in Ca++or in GMPCPP contain filaments whose dimensions indicate that they could be equivalent to the ‘thick’-filaments in sheets. Some tubes are hollow but others are filled by additional protein density. Helical FtsZ tubes differ from eukaryotic microtubules in that the filaments curve around the filament axis with a pitch of ~ 430 Å for Ca++-induced tubes or 590–620 Å for GMPCPP. However, their assemblyin vitroas well-ordered polymers over distances comparable to the inner circumference of a bacterium may indicate a rolein vivo. Their size and stability make them suitable for use in motility assays.
Sec61p Is the Main Ribosome Receptor in the Endoplasmic Reticulum of Saccharomyces cerevisiae
Authors: Prinz Anke , Hartmann Enno , Kalies Kai-Uwe
Published: October 2004

A characteristic feature of the co-translational protein
translocation into the endoplasmic reticulum (ER) is
the tight association of the translating ribosomes with
the translocation sites in the membrane. Biochemical
analyses identified the Sec61 complex as the main
ribosome receptor in the ER of mammalian cells. Similar
experiments using purified homologues from the
yeast Saccharomyces cerevisiae, the Sec61p complex
and the Ssh1p complex, respectively, demonstrated
that they bind ribosomes with an affinity similar to that
of the mammalian Sec61 complex. However, these
studies did not exclude the presence of other proteins
that may form abundant ribosome binding sites in the
yeast ER. We now show here that similar to the situation
found in mammals in the yeast Saccharomyces
cerevisiae the two Sec61-homologues Sec61p and
Ssh1p are essential for the formation of high-affinity
ribosome binding sites in the ER membrane. The number
of binding sites formed by Ssh1p under standard
growth conditions is at least 4 times less than those
formed by Sec61p.
Noncovalent Binding of Poly(ADP-Ribose) to Nuclear Matrix Proteins: Developmental Changes and Tissue Specificity
Authors: Malanga M. , Farina B.
Published: October 2004

Poly(ADP-ribose) is a nuclear polynucleotide involved in the regulation of chromatin functionsviacovalent and/or noncovalent modification of nuclear proteins. Using a binding assay on protein blots, we searched for poly(ADP-ribose) binding proteins in nuclear matrices from testes of differently aged rats as well as from various adult rat tissues (brain, liver, spleen). We found that nuclear matrix proteins represent a significant subset of the nuclear proteins that can establish noncovalent interactions with poly(ADP-ribose). The profiles of poly(ADP-ribose) binding nuclear matrix proteins appeared to be tissue-specific and changed during postnatal development in the testis.

The isolation and analysis of endogenous poly (ADP-ribose) from rat testes showed that the ADP-ribose polymers that bind nuclear matrix proteinsin vitroare also present under physiologic conditionsin vivo. These results further substantiate the possibility that poly(ADP-ribose) may affect chromatin functions through noncovalent interaction with specific protein targets, including nuclear matrix components.
Cloning, Heterologous Expression and Kinetic Analysis of Glycerol Kinase (TbGLK1) from Trypanosoma brucei
Authors: Steinborn K. , Szallies A. , Mecke D. , Duszenko M.
Published: October 2004

We have cloned and sequenced the gene for the glycerol kinase ofTrypanosoma brucei(TbGLK1), obtained by RT-PCR. The corresponding mRNA is 2.3 kb in size and contains an ORF encoding a protein with high homology to known glycerol kinases of other organisms. It is 512 amino acids in length with a PTS1-like targeting sequence (AKL) at its C-terminus, suggesting glycosomal compartmentalization of this enzyme. Although Northern blot analysis revealed higher mRNA levels in slender bloodstream forms than in the procyclic insect forms, specific glycerol kinase activities were found to be virtually identical in both life stages. Southern blot analysis suggested a single copy gene, but we were able to clone two alleles utmost similar to each other. Heterologous expression of the trypanosomal glycerol kinase inE. colienabled us to perform a kinetic analysis of this enzyme. In particular, we have been able to monitor ATP production from glycerol-3-phosphate and ADP, a reaction which, although thermodynamically very unfavorable, is regarded essential for the survival ofTrypanosoma bruceiunder anoxic conditions. Since the unique spatial separation of glycolysis in the kinetoplastida imposes important consequences for the regulation of the energy metabolism in these organisms, we discuss the observed differences between TbGLK1 and glycerol kinases from other organisms in view of its physiological relevance.