bluuebunny/crossref_metadata_2025 huggingface dataset:
Nanosecond electron transfer kinetics in photosystem I as obtained from transient EPR at room temperature
Authors: Bock Christian H. , van der Est Arthur J. , Brettel Klaus , Stehlik Dietmar
Published: July 2002

Transient EPR spectra of photosystem I (PS I) in spinach chloroplasts and PS I particles prepared fromSynechococcusare presented. Two consecutive spectra are observed after the laser pulse. The decay time of the first spectrum is equal to the rise time of the second spectrum. The two spectra represent sequential charge‐separated states in the electron transfer chain and the time constant of the electron transfer step between them is found to bet1/e= 260 ± 20 ns for theSynechococcusPS I particles as well as for the spinach chloroplasts. The first spectrum is assigned to the P+700A−1pair, where A−1is the second electron acceptor, probably a quinone‐like molecule. The second spectrum covers the region of the P+700signal and may be part of the spectrum due to the coupled radical pair P+700(FeS), where (FeS)−is the first iron‐sulfur center along the electron transfer chain.
In vitro transcription of the mouse whey acidic protein promoter is affected by upstream sequences
Authors: Lubon Henryk , Pittius Christoph W. , Hennighausen Lothar
Published: July 2002

The promoter sequences from −175 to +10 of the mouse whey acidic protein (WAP) gene are a target for sequence‐specific binding of nuclear proteins [(1987) Nucleic Acids Res. 15, 2103–2121]. Using in vitro transcription assays based on nuclear extracts, transcription factors were shown to bind to these sequences and promoter upstream sequences were found to stimulate transcription.
Phorbol esters stimulate a phospholipase D‐catalysed reaction with both ester‐ and ether‐linked phospholipids in HeLa cells
Authors: Hii Charles S.T. , Kokke Yasmin S. , Pruimboom Wanda , Murray Andrew W.
Published: July 2002

Phorbol ester; Phospholipase D; Ether phospholipid
Arachidonic acid evokes inositol phospholipid hydrolysis in astrocytes
Authors: Murphy Sean , Welk Greg
Published: July 2002

Arachidonate; Inositol phosphate; Astrocyte; Phospholipase C; Receptor; Cell signaling
Photoactivation of rhodopsin and interaction with transducin in detergent micelles
Authors: König Bernd , Welte Wolfram , Hofmann Klaus Peter
Published: July 2002

Rhodopsin; Metarhodopsin II; Transducin; G‐protein; (Detergent micelle)
Photolabile μ‐conotoxins with a chromogenic phenyldiazirine
Authors: Hatanaka Yasumaru , Yoshida Eiichi , Nakayama Hitoshi , Abe Teruo , Satake Mei , Kanaoka Yuichi
Published: July 2002

Three photoreactive derivatives of μ‐conotoxin G IIIA have been prepared as photoaffinity labeling reagents for muscle‐type sodium channels. The reagents competitively inhibited the binding of saxitoxin to the eel sodium channel with K
i values of 11–18 nM. The introduced chromogenic phenyldiazirine group on the toxin was photolyzed efficiently, and spectroscopic properties of the reagents demonstrated that irradiation and detection can be performed in a spectral region where the absorptions due to most of biological macromolecules are negligible.
Cyclic changes in translation of the mitochondrial isoenzyme account for the rhythm of aspartate aminotransferase activity in Leptosphaeria michotii
Authors: Romestant M. , Jerebzoff-Quintin S. , Jerebzoff S.
Published: July 2002

Aspartate aminotransferase in Leptosphaeria michotii has previously been shown to have an activity rhythm in constant conditions. The enzyme is present as two isofonns whose levels were quantified along the activity rhythm by ELISA, using specific polyclonal antibodies raised against polypeptides purified to a state of apparent homogeneity. The time‐course of the level of the cytosolic isoform remained unchanged along the experiment. On the contrary, the cyclic variations in amount and transaminase activity (using cysteine sulfinate as substrate) of the mitochondrial isofonn gave rise to the aspartate aminotransferase activity rhythm of the fungus. The mRNA levels of the two isofonns, as determined by in vitro heterologous translation, remained monotonous along the daily cycle. These results and the sensitivity of the rhythm towards protein synthesis inhibitors are consistent with the hypothesis that the aspartate aminotransferase activity rhythm in this species is caused by some mechanism controlling the efficiency of translation of mitochondrial isoform mRNA.
Two novel brain proteins, CaBP33 and CaBP37, are calcium‐dependent phospholipid‐ and membrane‐binding proteins
Authors: Donato Rosario , Giambanco Ileana , Pula Grazia , Bianchi Roberta
Published: July 2002

Two acidic Ca2+ ‐binding proteins (CaBP33 and CaBP37) purified from bovine brain have been characterized in terms of immunological properties, heat‐sensitivity, electrophoretic mobility, and Ca2+‐dependent binding to negatively charged phospholipids and to brain membranes. They were induced to bind to membranes by homogenization of brain tissue in the presence of CaCl2. The membrane‐bound CaBP33/CaBP37 mixture resisted extraction with detergents and was solubilized with high concentrations of EGTA/KC1. However, apparent Ca2+‐independent binding of the two proteins to membranes seemed to occur as well. This latter fraction of membrane‐bound CaBP33 and CaBP37 could be solubilized with Triton X‐100, indicating that brain membranes normally contain the two proteins as intrinsic components.
De novo protein synthesis is essential to human interferon γ gene expression by the stimulation with polyI:polyC
Authors: Tamura-Nishimura Motoko , Sasakawa Shigeru
Published: July 2002

Transcription of human interferon (IFN) γ gene is induced in human peripheral lymphocyte nylon‐nonadherent cells (NNA cells) by double strand RNA poly I:poly C [(1985) J. Interferon Res. 5, 77‐84]. In this report, the necessity of de novo protein synthesis in an early stage of IFN γ gene expression is described. For induction of IFN γ gene expression, only initial 4 h treatment of poly I:poly C to NNA cells is sufficient. Addition of inhibitor of protein synthesis, cycloheximide (CHX), at an early stage of induction periods (0–4 h) inhibits the IFN γ induction by poly I:poly C. Cell free translation assay using RNAs isolated from NNA cells which are induced by poly I:poly C in the presence of CHX reveals that in these RNAs, IFN γ mRNA does not exist. These results demonstrate that CHX inhibits de novo synthesis of a certain protein (or proteins) and for lack of the protein(s), IFN γ mRNA cannot be transcribed. The evidence is also described in this report which suggests that the essential protein(s) might be that (those) involved in protein kinase C (pkC) activation.
Rapid increase in inositol pentakisphosphate accumulation by nicotine in cultured adrenal chromaffin cells
Authors: Sasakawa Nobuyuki , Nakaki Toshio , Kato Ryuichi
Published: July 2002

When [3H]inositol‐prelabeled cultured bovine adrenal chromaffin cells were stimulated with nicotine (10 μM), a large and transient increase in [3h]‐inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached the maximum level at 15 s, then declined to the basal level at 2 min. Nicotine also induced [3H]inositol tetrakisphosphate (InsP4) and [3H]inositol hexakisphosphate (InsP6) accumulation with a slower time course and a lesser magnitude than [3HInsP5. The peaks of [3H]InsP4, [3HInsP5 and 3H]InsP6 coincided with those of 32P radioactivity, when cells were doubly labeled with [3H]inositol and inorganic 32P. These results suggest that inositol pentakisphosphate is rapidly increased by nicotine, a cholinergic agonist, in cultured adrenal chromaffin cells.
Binding of GTP to transducin is not inhibited by arrestin and phosphorylated rhodopsin
Authors: Fukada Yoshitaka , Yoshizawa Tôru , Saito Tetsuya , Ohguro Hiroshi , Akino Toyoaki
Published: July 2002

In the presence of a photobleaching intermediate of unphosphorylated or phosphorylated rhodopsin (Rh∗), the binding of GppNHp to transducin was measured with or without arrestin for elucidation of the shut‐off mechanism of the visual transduction process in bovine rod outer segments. The ability of Rh∗to catalyze the formation of the transducin‐GppNHp complex in the absence of arrestin was independent of the degree of phosphorylation of Rh∗. Furthermore, the catalyzing ability of the phosphorylated Rh∗was not reduced by the addition of arrestin. These observations indicate that the interaction between phosphorylated Rh∗and transducin was not inhibited by arrestin. Thus, the hypothesis was not supported that the PDE shut‐off process is a simple competition between transducin and arrestin for binding to phosphorylated Rh∗.
Evolutionary specificity of hydrins, new hydroosmotic neuropeptides: occurrence of hydrin 2 (vasotocinyl‐Gly) in the toad Bufo marinus but not in the viper Vipera aspis
Authors: Michel G. , Rouillé Y. , Chauvet M.T. , Chauvet J. , Acher R.
Published: July 2002

Hydrin 2 (vasotocinyl‐Gly), a hydroosmotic peptide resulting from differential processing of provasotocin and recently identified in frog neurohypophysis, has been looked for in the pituitary gland of an exotic toad (Bufo marinus) and of a reptile (Vipera aspis). Hydrin 2 has been found in the amphibian but not in the reptile. This result confirms the evolutionary specificity of hydrin 2 that has been identified only in frogs and toads but not in birds and reptiles. Occurrence of hydrin 2 is explained by its regulatory function on the water permeability of the skin of anurans.
X‐ray crystal structure of sangivamycin, a potent inhibitor of protein kinases
Authors: Lebioda Lukasz , Hargrave Paul A , Palczewski Krzysztof
Published: July 2002

The X‐ray crystal structure of sangivamycin, a potent nucleoside inhibitor of protein kinases, has been determined. Sangivamycin crystallizes from water with its purine ring in a conformation anti to its ribose sugar. Such an anti conformation has been detected in solution for sangivamycin and other potent protein kinase inhibitors and appears to correlate with inhibitor potency [(1990) Biochemistry (in press)]. An intramolecular hydrogen bond between purine ring substituents is detected in the X‐ray structure and may be an important structural feature of sangivamycin related to its degree of inhibition of rhodopsin kinase and of protein kinases C and A.
Light‐harvesting complexes of brown algae Biochemical characterization and immunological relationships
Authors: Passaquet C. , Thomas J.C. , Caron L. , Hauswirth N. , Puel F. , Berkuloff C.
Published: July 2002

The pigment composition of the light‐harvesting complexes isolated from several brown algae belonging to different orders has been analysed by reverse‐phase HPLC. Relative to whole chloroplasts, they were markedly enriched in Chlc, fucoxanthin and violaxanthin and conversely depleted in Chla. The relative molar proportions of the 4 main pigments (Chla/Chle/fucoxanthin/violaxanthin) ranged from 100:18:76:6 to 100:30:107:17. The protein moiety of LH complexes of all the species studied were composed of one or two main polypeptide components in the range of 19–22 kDa. Those polypeptide subunits were arranged in polymeric particles about 240 kDa inLaminaria saccharina. A polyclonal antibody raised against the LH polypeptide ofFucus serratushas been tested on LH apoproteins of other Chromophytes and Chlorophytes. Phylogenic inplications of these results discussed.
Mutating P2 and P1 residues at cleavage junctions in the HIV‐1 pol polyprotein Effects on hydrolysis by HIV‐1 proteinase
Authors: Jupp Ray A. , Phylip Lowri H. , Mills John S. , Le Grice Stuart F.J. , Kay John
Published: July 2002

Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (‐Leu*Phe‐) and (b) between the p51 and RNase H domain (‐Phe*Tyr‐) within p66 of RT in the HIV‐1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a β‐branched (He) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a β‐branched (Val) residue in the P1 position flanking such ‐Hydrophobic*Hydrophobic‐ junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.
Chemical synthesis of 10 kDa chaperonin Biological activity suggests chaperonins do not require other molecular chaperones
Authors: Mascagni Paolo , Tonolo Mirta , Ball Haydn , Lim Marguerita , Ellis R.John , Coates Anthony
Published: July 2002

Molecular chaperones are required for the correct folding and assembly of certain other polypeptides. It is not known whether molecular chaperones themselves require other chaperones to become functional. A 97‐amino acid chaperone, the chaperonin 10 protein was chemically synthesised so that during synthesis and purification there was no contact of the chaperone with any other protein. The purified, synthetic chaperonin 10 protein formed oligomeric structures spontaneously and was biologically active as a chaperonin. This is the first description of a chemically synthesised chaperonin, and suggests that no other chaperones are required for correct folding, polymerisation and biological activity of this chaperone.
Thegagprecursor contains a specific HIV‐1 protease cleavage site between the NC (P7) and P1 proteins
Authors: Wondrak Ewald M. , Louis John M. , de Rocquigny Hugues , Chermann Jean-Claude , Roques Bernard P.
Published: July 2002

The predicted protease cleavage site (p7/pl; [J. Virol. 66 (1992) 1856‐1865]) within the nucleocapsid precursor protein (p15) of human immunodeficiency virus, type 1, was confirmed using an in vitro assay employing recombinant HIV‐1 protease and a chemically synthesized 72 amino acid polypeptide containing the p7 and p1 protein domains of the nativegagpolyprotein. The cleavage occurred between amino acid 55 (N) and amino acid 56 (F) of the polypeptide, as determined by N‐terminal sequencing. The hydrolysis was optimal at pH 6.0 and at high salt concentration. The kinetic parametersKm,kcatandKcat/Kmwere 99 μM (±8), 0.152 s−1(± 0.002) and 1.56 mM−1· s−1(± 0.11), respectively. Reconstituted as well as denatured polypeptides were cleaved at approximately the same rate, demonstrating that the conformation of the p7 protein, as a result of the Zn2+‐binding, had no significant effect on the rate of hydrolysis of the p7/pl cleavage.
The secondary structure of bacteriorhodopsin in organic solution
Authors: Torres Jaume , Padrós Esteve
Published: July 2002

Fourier transform infrared spectroscopy is used to estimate the secondary structure of bacteriorhodopsin dissolved in Chloroform‐methanol (1:1 v/v), 0.1 M LiClO4. Curve‐fitting of the deconvolved spectra in the amide I region shows that the total content of α‐helices, reverse turns and β‐sheets are similar to the native state. However, the αII‐helices, which are the major helical class in native bacteriorhodopsin, are greatly decreased in the solubilized sample. Similarly, the reverse turns and the β‐sheets are strongly altered.
Non‐covalent binding of the heavy atom compound [Au(CN)2]− at the halide binding site of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10
Authors: Verschueren K.H.G. , Franken S.M. , Rozeboom H.J. , Kalk K.H. , Dijkstra B.W.
Published: July 2002

The Na[Au(CN)2] heavy atom derivative contributed considerably to the successful elucidation of the crystal structure of haloalkane dehalogenase isolated from Xanthobacter autotrophicus GJ10. The gold cyanide was located in an internal cavity of the enzyme, which also contains the catalytic residues. Refinement of the dehalogenase‐gold cyanide complex at 0.25 nm to an R‐factor of 16.7% demonstrates that the heavy atom molecule binds non‐covalently between two tryptophan residues pointing into the active site cavity. At this same site also chloride ions can be bound. Therefore, inhibition of dehalogenase activity by the Au(CN)2
− presumably occurs by competition for the same binding site as substrates.
Crystal structure of a distal site double mutant of sperm whale myoglobin at 1.6 Å resolution
Authors: Rizzi Menico , Bolognesi Martino , Coda Alessandro , Cutruzzolà Francesca , Allocatelli Carlo Travaglini , Brancaccio Andrea , Brunori Maurizio
Published: July 2002

The three‐dimensional structure of sperm whale myoglobin His64(E7)→Val,Thr64(E10)→Arg double mutant has been studied by X‐ray crystallography at 1.6 Å resolution, and refined to a crystallographicR‐factor of 0.197. The Arg67(E10) side chain is extended in the direction of the ligand binding site, and its NH1 atom is at a distance of 3.11 Å from the NH1 atom of Arg45(CD3), which is also pointing towards the distal site. Both are kept in this position by hydrogen bonding and electrostatic interactions with a solvent sulfate ion, located amongst the two, on the protein surface. No liganded water molecule is present at the sixth coordination position of the Fe(III) heme.
Photosynthetic oxygen evolution: Net charge transients as inferred from electrochromic bandshifts are independent of proton release into the medium
Authors: Haumann Michael , Bögershausen Oliver , Junge Wolfgang
Published: July 2002

The manganese containing center of the oxygen evolving complex accumulates four oxidizing equivalents in the four stepped water oxidation cycle. Based on experiments on electrochromic absorption transients and the reduction rate of the primary electron donor, P680, it has been speculated that the oscillations of these variables reflect the net charge of the center as calculated from the difference between electron abstraction and proton release into the medium. We compared proton release with electrochromism in thylakoids and core particles, and under variation of the rate of proton release. We found no equivalent of the variations of the extents and the rates of proton release in electrochromism. The oscillatory pattern of the latter reflects the topological properties of the stepped charge storage relative to the position and orientation of electrochromically responsive pigments rather than responding to proton release from the periphery.
Mechanisms of stimulus‐evoked intracellular acidification in frog nerve fibres
Authors: Khodorov B. , Valkina O. , Turovetsky V.
Published: July 2002

Measurements of cytoplasmic pH (pHi) in frog nerve fibers (sciatic nerve and its thin bundles) were performed by using fluorescein diacetate. Earlier it had been established that veratridine (VER) treatment of the nerve greatly enhances the stimulus‐evoked intracellular acidification (SEIA) which becomes irreversible after blockade of the Na+/K+ pump with ouabain. Present experiments have shown that inhibition of lactic acid production by iodacetamide (5 mM) or blockade of Cl− influx by SITS do not prevent or attenuate the VER‐ and stimulus‐evoked decrease in pHi. Blockade of Na+/H+ exchange by EIPA impedes pHi recovery following repetitive stimulation. Lowering of external pH (pHe) to 6.5 enhances, while elevation of pHe to 9.5 greatly diminishes SEIA, both in the presence or absence of VER. The hypothesis is put forward that SEIA results from excessive influx of H+ and Na+ into the fiber via activated Na+ channels: internal Na+ suppresses Na+/H+ exchange which potentiates the pHi decrease caused by H+ influx.
Light‐induced Fourier transform infrared spectrum of the cation radical P680+
Authors: Allakhverdiev S.I. , Ahmed A. , Tajmir-Riahi H.-A. , Klimov V.V. , Carpentier R.
Published: July 2002

The structure of the primary electron donor of photosystem II, P680, is still under debate. It is not decided if it is composed of a chlorophyll (Chl) monomer or dimer. In this study, Fourier transform infrared (FTIR) spectroscopy was used to analyze the changes in the vibration modes occurring upon photooxidation of P680 in a Mn‐depleted PS II preparation. It is demonstrated that illumination of the above in the presence of artificial electron acceptors results in a light‐minus‐dark absorbance change typical of the formation ofP680+. The light‐minus‐dark difference FTIR spectrum obtained under similar conditions is characterized by two negative peaks located at 1694 and 1652 or 1626 cm−1that can be assigned to the 9‐keto groups of the P680 Chl, the latter band being indicative of a strongly associated group. These vibrations are shifted to 1714 and 1676 cm−1, respectively, in the positive features of the difference spectrum attributed to P680+. The occurrence of two pairs of bands attributed to 9‐keto groups is discussed in terms of P680 being formed of a Chl dimer.
Magnesium and calcium concentration in the pregnant and non‐pregnant myometrium
Authors: Cunze T. , Rath W. , Osmers R. , Martin M. , Warneke G. , Kuhn W.
Published: July 2002

Objectives: To investigate the effect of labor on the magnesium and calcium concentrations in the human myometrium and to compare them with the concentrations in the non‐pregnant uterus. Method: Samples of the isthmic part of the uterus were taken from 42 patients not in labor, 31 patients in regular labor during a cesarean section, and 37 premenopausal patients on whom a hysterectomy was performed. Results: (i) The magnesium concentration dropped significantly during labor; (ii) the calcium concentration in the tissue showed no significant changes during labor; (iii) the magnesium and calcium concentrations were significantly higher during pregnancy; (iv) the distribution of electrolytes in the myometrium differed significantly in the corpus, isthmus and cervix uteri. Conclusions: The electrolyte concentration in the myometrium depends on the place of removal. A low magnesium concentration in the pregnant human myometrium could be a cause of premature labor.
NMDA receptors in mouse barrel cortex during normal development and following vibrissectomy
Authors: Głażewski S. , Kossut M. , Skangiel‐Kramska J.
Published: July 2002

The development of N‐methyl‐d‐aspartate (NMDA) receptors and the effects of vibrissectomy upon [3H]MK‐801 binding were examined in the barrel cortex of mice. Autoradiograhic studies showed that initially very low binding of [3H]MK‐801 sharply increased during the second postnatal week reaching the adult level by the end of the third week. Scatchard analysis performed on cortical membrane preparations indicated that this rise of [3H]MK‐801 labelling was due to an increase in the number of binding sites and a decrease of Kd at postnatal day 15 and 28. The interlaminar differences of labelling were registered from postnatal day 8. Changes of interlaminar distribution were found during the second and third postnatal weeks. In adult barrel cortex the highest binding was found in supragranular layers. In layer IV of the cortex, the pattern of binding resembled the pattern of barrels. Unilateral denervation of vibrissae performed in neonatal and adult mice did not alter the intensity of [3H]MK‐801 labelling or the laminar distribution of binding sites. These results suggest that NMDA receptor binding does not reflect the plastic changes occurring in the barrel cortex.
Maternal infectious diseases during pregnancy and obstetric complications in the etiology of distinct subtypes of schizophrenia: further evidence from maternal hospital records
Authors: Franzek E , Stöber G
Published: July 2002

On the basis of 24 maternity hospital records, the current study investigated the validity of maternal recall and the relationship of maternal infections during pregnancy and obstetric complications (OCs) to different diagnostic subgroups of endogenous psychoses on which we reported previously in this journal. Maternal recall showed good agreement to maternity hospital records in the Lewis and Murray scale (ϑ = 0.74). With regard to infectious diseases during pregnancy maternal recall and records showed a weaker, but also good correlation (ϑ = 0.18). Psychoses with low genetic loading had more OCs than psychoses with high genetic loading. Maternal infectious diseases, especially during the fourth or fifth month of gestation, were significantly allocated to Leonhard's systematic schizophrenias. Data from maternity hospital records support our report that infectious diseases during midgestation and further perinatal complications seem to be important etiologic factors in systematic forms of schizophrenia without marked familial loading.
DrosophilaMTN: a metazoan copper‐thionein related to fungal forms
Authors: Valls M. , Bofill R. , Romero-Isart N. , Gonzàlez-Duarte R. , Abián J. , Carrascal M. , Gonzàlez-Duarte P. , Capdevila M. , Atrian S.
Published: July 2002

TwoDrosophilametallothioneins (MT) have been reported: MTN, a 40 residue peptide including 10 Cys, and MTO, a 43 residue peptide including 12 Cys. However, neither functional nor evolutionary analyses for either of theDrosophilaMT are available. Here, heterologous expression ofMtninEscherichia coliis reported. The metal binding abilities of the Cu‐ and Zn‐MTN complexes conformed in vivo, as well as the features of the Cd‐ and Cu‐aggregates produced by metal replacement in vitro, have been determined by atomic emission spectrometry, circular dichroism and electrospray ionization mass spectrometry. Primary structure relationships with other MT have been examined. The results indicate a close resemblance of MTN to fungal copper‐thioneins.
Depletion of phosphatidylethanolamine affects secretion of Escherichia coli alkaline phosphatase and its transcriptional expression
Authors: Mikhaleva Natalia I , Golovastov Victor V , Zolov Sergey N , Bogdanov Mikhail V , Dowhan William , Nesmeyanova Marina A
Published: July 2002

In this report we demonstrate that depletion of the major phospholipid phosphatidylethanolamine, a single non‐bilayer forming phospholipid of Escherichia coli, significantly reduces the secretion efficiency of alkaline phosphatase in vivo. Secretion, however, is correlated with the content in membranes of cardiolipin, which in combination with selected divalent cations has a strong tendency to adopt a non‐bilayer state indicating the possible involvement of lipid polymorphism in efficient protein secretion. Depletion of this zwitterionic phospholipid also inhibits expression of the protein controlled by the endogenous PPHO promoter but not the PBAD promoter, which is suggested to be due to the effect of unbalanced phospholipid composition on the orthophosphate signal transduction system (Pho regulon) through an effect on its membrane bound sensor.
The role of PDR13 in tolerance to high copper stress in budding yeast
Authors: Kim Do-Young , Song Won-Yong , Yang Young-Yell , Lee Youngsook
Published: July 2002

PDR13inSaccharomyces cerevisiaecontributes to drug resistance via sequential activation ofPDR1andPDR5. In this study, we found that aPDR13deletion mutant was hypersensitive to Cu2+compared to the wild‐type counterpart. The Cu2+tolerance mechanism mediated by Pdr13 does not seem to involve Pdr1 or Pdr5, since mutants harboring a deletion of either thePDR1orPDR5gene did not show elevated Cu2+sensitivity. Instead, we found that thePDR13null mutant could not expressCUP1orCRS5metallothionein at wild‐type levels when subjected to high Cu2+stress. These results suggest that Pdr13 contributes to high Cu2+tolerance ofS. cerevisiae, at least in part, via a mechanism involving metallothionein expression.
Similarity of bacteriorhodopsin structural changes triggered by chromophore removal and light‐driven proton transport
Authors: Ludlam Gary J. , Rothschild Kenneth J.
Published: July 2002

Bacteriorhodopsin (bR) is the light‐driven proton pump found in the purple membrane ofHalobacterium salinarium. A series of conformational changes occur during the bR photocycle which involve alterations in buried‐helical structure as well as in the protonation state of Asp residues which are part of the proton transport pathway. Here we report evidence that similar conformational changes occur upon removal of the retinylidene chromophore of bacteriorhodopsin to form the apoprotein bacterioopsin (bO). This suggests a simple ligand‐binding model of proton transport in bacteriorhodopsin which may have relevance to other transport and signal transducing membrane proteins including the visual photoreceptor rhodopsin.
Role of inward K+ channel located at carrot plasma membrane in signal cross‐talking of cAMP with Ca2+ cascade
Authors: Kurosaki Fumiya
Published: July 2002

Treatment of cultured carrot cells with dibutyryl cAMP or forskolin resulted in the appreciable decrease in extracellular K+ concentration. This decrease was found to be transient and the concentration of the ion in the culture medium restored to the original level within few minutes. The cAMP‐induced decrease in K+ level in the medium was almost completely inhibited when carrot cells were incubated in the presence of K+ channel blockers, CsCl and tetraethylammonium chloride. Appreciable amounts of 45Ca2+ were discharged from 45Ca2+‐loaded inside‐out vesicles of carrot plasma membrane by the stimulation with cAMP, however, the release of the ion was significantly inhibited in the presence of the K+ channel blockers. The release of 45Ca2+ from the vesicles was also observed when K+ current was evoked with an ionophore, valinomycin, even in the absence of cAMP. These results suggest that the gating of some of the inward K+ channels located at plasma membrane of cultured carrot cells is controlled by cytoplasmic concentration of cAMP and the inward K+ current across the plasma membrane induced by the nucleotide elicits Ca2+ influx into the cells possibly by the activation of voltage‐dependent Ca2+ channels.
MARCKS, a major protein kinase C substrate, assumes non‐helical conformations both in solution and in complex with Ca2+‐calmodulin
Authors: Matsubara Mamoru , Yamauchi Emiko , Hayashi Nobuhiro , Taniguchi Hisaaki
Published: July 2002

MARCKS, a major cellular substrate for protein kinase C, plays important roles in various cellular functions and its functions are regulated by calmodulin. We have studied the conformational properties of recombinant human MARCKS in solution and in complex with calmodulin. Circular dichroism (CD) spectra showed a high content of random coil in physiological solution. When MARCKS or MARCKS‐derived calmodulin‐binding peptide was complexed with Ca2+‐calmodulin, little change was observed in the CD spectra, suggesting that MARCKS binds with calmodulin in a non‐helical conformation, which is unique among the calmodulin‐binding proteins.
Purification and characterisation of an insulin‐stimulated protein‐serine kinase which phosphorylates acetyl‐CoA carboxylase
Authors: Heesom K.J , Moule S.K , Denton R.M
Published: July 2002

An insulin‐stimulated protein kinase specific for acetyl‐CoA carboxylase has been purified from rat epididymal adipose tissue using Mono‐Q chromatography. The kinase binds to (and phosphorylates) the relatively inactive, dimeric form of acetyl‐CoA carboxylase, but not to its active, polymeric form, and this property has been used to purify the kinase. Under the conditions used, phosphorylation by the purified kinase did not result in a detectable increase in acetyl‐CoA carboxylase activity. These studies also led to the recognition of an `activator' protein which is capable of increasing the activity of acetyl‐CoA carboxylase without changing its phosphorylation state. It is suggested that this `activator' protein, together with the insulin‐activated acetyl‐CoA carboxylase kinase, may play a role in the activation of acetyl‐CoA carboxylase by insulin.
A de novo missense mutation of human cardiac Na+ channel exhibiting novel molecular mechanisms of long QT syndrome
Authors: Makita Naomasa , Shirai Nobumasa , Nagashima Masato , Matsuoka Rumiko , Yamada Yoichi , Tohse Noritsugu , Kitabatake Akira
Published: July 2002

Mutations in a human cardiac Na+ channel gene (SCN5A) are responsible for chromosome 3‐linked congenital long QT syndrome (LQT3). Here we characterized a de novo missense mutation (R1623Q, S4 segment of domain 4) identified in an infant Japanese girl with a severe form of LQT3. When expressed in oocytes, mutant Na+ channels exhibited only minor abnormalities in channel activation, but in contrast to three previously characterized LQT3 mutations, had significantly delayed macroscopic inactivation. Single channel analysis revealed that R1623Q channels have significantly prolonged open times with bursting behavior, suggesting a novel mechanism of pathophysiology in Na+ channel‐linked long QT syndrome.
Sulfitolysis and thioredoxin‐dependent reduction reveal the presence of a structural disulfide bridge in spinach chloroplast fructose‐1,6‐bisphosphatase
Authors: Drescher Dorothee F , Follmann Hartmut , Häberlein Ingo
Published: July 2002

A significant difference between cytosolic and chloroplastic fructose‐1,6‐bisphosphatase (FbPase) is an extra peptide in the middle of chloroplast FbPase which contains three additional cysteine residues. Sit‐directed mutagenesis experiments have shown that at least two of these cysteine residues are involved in forming the regulatory disulfide bridge [Jacquot, J.‐P. et al., FEBS Lett. 401 (1997) 143–147] which is the presupposition for the thioredoxin‐dependent control of chloroplast FbPase activity. Here we report that each subunit of the FbPase contains an additional structural disulfide bridge which has been observed by combined application of thioredoxins and sulfitolysis. Observation of the structural disulfide bridges by sulfitolysis was only possible when the FbPase was already specifically reduced by the homologous thioredoxin species TRm and TRf from spinach chloroplasts. Interestingly, the accessibility of the structural disulfide bridge for sulfite ions depends on the thioredoxin species engaged in the thioredoxin/FbPase complex.
Site‐directed mutagenesis and chemical modification of the two cysteine residues of the UDP‐N‐acetylmuramoyl:l‐alanine ligase of Escherichia coli
Authors: Nosal Florence , Masson Anne , Legrand Raymond , Blanot Didier , Schoot Bernard , van Heijenoort Jean , Parquet Claudine
Published: July 2002

Site‐directed mutagenesis and chemical modification of the two cysteine residues of the MurC l‐alanine‐adding enzyme from Escherichia coli were undertaken to study their possible role in activity and stability. Their replacement by alanine was not critical for activity. However, C230 played a role in enzyme stability and substrate binding. N‐Ethylmaleimide alkylation led to monoalkylated and dialkylated proteins. The monoalkylated protein had mostly unmodified C230 residues. The extent of alkylation of C230 paralleled the loss of activity, whereas that of C426 did not. Protection against inactivation by β,γ‐imidoadenosine 5′‐triphosphate implied the involvement of C230 in the ATP binding site.
2D‐isolation of pure plasma and thylakoid membranes from the cyanobacteriumSynechocystissp. PCC 6803
Authors: Norling Birgitta , Zak Elena , Andersson Bertil , Pakrasi Himadri
Published: July 2002

Aqueous polymer two‐phase partitioning in combination with sucrose density centrifugation offered, for the first time, a 2D‐separation method for the isolation of pure plasma and thylakoid membranes from the cyanobacteriumSynechocystis6803 without any cross‐contaminations. The purity of the membrane fractions was verified by immunoblot analysis using antibodies against membrane‐specific marker proteins. As an initiation of a proteomics project, two prominent proteins, which were observed only in the plasma membrane (Slr1513, a hypothetical protein, and HofG, a general secretion pathway protein), or in the thylakoid membrane (PsaE, a photosystem I protein, and NdhH, a subunit of NADH dehydrogenase), were identified.
Cytosolic lysophosphatidylcholine/transacylase in the production of dipolyunsaturated phosphatidylcholine in bonito muscle
Authors: Hirano Kaoru , Ito Tetsuya , Morihara Hisanori , Tanaka Tamotsu , Satouchi Kiyoshi
Published: July 2002

Phosphatidylcholine with docosahexaenoic acid at both sn‐1 and sn‐2 positions occurs in relatively high abundance in bonito muscle. To explore a possible route for the dipolyunsaturated molecular species, phosphatidylcholine formation from 2‐[1‐14C]linoleoyl lysophosphatidylcholine was examined using a cytosolic fraction from bonito muscle. The formation of radiolabeled phosphatidylcholine was greatest at 15°C and did not require the presence of cofactors such as CoA and calcium. By DEAE‐cellulofine column chromatography, the activity to form phosphatidylcholine was separated from that of phospholipase A1, and the specific activity increased by about 100‐fold. The possible involvement of cytosolic lysophosphatidylcholine/transacylase in synthesis of dipolyunsaturated phosphatidylcholine is discussed.
NMR spatial structure of α‐conotoxin ImI reveals a common scaffold in snail and snake toxins recognizing neuronal nicotinic acetylcholine receptors1
Authors: Maslennikov Innokenty V , Shenkarev Zakhar O , Zhmak Maxim N , Ivanov Vadim T , Methfessel Christoph , Tsetlin Victor I , Arseniev Alexander S
Published: July 2002

A 600 MHz NMR study of α‐conotoxin ImI fromConus imperialis, targeting the α7 neuronal nicotinic acetylcholine receptor (nAChR), is presented. ImI backbone spatial structure is well defined basing on the NOEs, spin‐spin coupling constants, and amide protons hydrogen‐deuterium exchange data: rmsd of the backbone atom coordinates at the 2–12 region is 0.28 Å in the 20 best structures. The structure is described as a type I β‐turn (positions 2–5) followed bya distorted helix (positions 5–11). Similar structural psattern can be found in all neuronal‐specific α‐conotoxins. Highly mobile side chains of the Asp‐5, Arg‐7 and Trp‐10 residues form a single site for ImI binding to the α7 receptor. When depicted with opposite directions of the polypeptide chains, the ImI helix and the tip of the central loop of long chain snake neurotoxins demonstrate a common scaffold and similar positioning of the functional side chains, both of these structural elements appearing essential for binding to the neuronal nAChRs.
Interaction of tumor and normal blood cells with ethylene oxide and propylene oxide block copolymers
Authors: Melik-Nubarov N.S. , Pomaz O.O. , Dorodnych T.Yu. , Badun G.A. , Ksenofontov A.L. , Schemchukova O.B. , Arzhakov S.A.
Published: July 2002

Ethylene oxide and propylene oxide block copolymers (pluronics) are widely known as agents that promote drug penetration across biological barriers. We have studied the interaction of normal and malignant blood cells with pluronics L61 and P85 that have different hydrophobicity. SP2/0 myeloma cells accumulated pluronics while normal cells adsorb most of the polymer on the surface. Interaction of pluronics with cells resulted in drastic changes of membrane microviscosity. Tumor cell membrane microviscosity decreased after pluronics adsorption, in contrast to normal cells, whose membrane microviscosity was enhanced. We suppose that sensitivity of tumor cell membrane microviscosity to the pluronics action correlates with its permeability for molecular substances.
Imaging fluorescence resonance energy transfer between two green fluorescent proteins in living yeast
Authors: Sagot Isabelle , Bonneu Marc , Balguerie Axelle , Aigle Michel
Published: July 2002

We show that fluorescence resonance energy transfer between two mutants of the green fluorescent protein (GFP) can be monitored by imaging microscopy in living yeast. This work is based on the constitutive expression of a GFP‐containing fusion protein and the inducible expression of the tobacco etch virus (TEV) protease. In the fusion protein, the P4.3 GFP mutant is linked to the YS65T GFP mutant by a spacer bearing the TEV protease‐specific cleavage site.
Anandamide stimulates phospholipase D activity in PC12 cells but not in NIH 3T3 fibroblasts
Authors: Kiss Zoltan
Published: July 2002

The endogenous cannabinoid arachidonoylethanolamide was previously reported to have no effects on the phospholipase D activity in Chinese hamster ovary cells expressing the human brain‐specific cannabinoid receptor, while in mouse peritoneal cells, Δ9‐tetrahydrocannabinol stimulated this enzyme. In this work, arachidonoylethanolamide (0.1–1 μM) was found to stimulate the phospholipase D‐mediated phospholipid hydrolysis in rat adrenal pheochromocytoma PC12 cells, but not in mouse NIH 3T3 fibroblasts. The phospholipase D‐activating effects of arachidonoylethanolamide were comparable to those elicited by phorbol ester and nerve growth factor, while arachidonic acid (1 μM) had no effects. The results show that, depending on the cell type, arachidonoylethanolamide can be an activator of the phospholipase D system.
A complex containing βTrCP recruits Ccd34 to catalyse ubiquitination of IκBα
Authors: Vuillard Laurent , Nicholson John , Hay Ronald T
Published: July 2002

Activation of transcription factor NF‐κB is accomplished by degradation of its inhibitor IκBα. Signal induced phosphorylation of IκBα on serine 32 and 36 targets the protein for ubiquitination on lysine 21 and 22. Here we use a phosphorylated peptide substrate representing residues 20–43 of IκBα to investigate requirements for ubiquitination of IκBα. Phosphorylation dependent polyubiquitination is carried out by a multiprotein complex containing βTrCP, Skp1 and Cdc53 (Cul1). In the presence of ubiquitin activating enzyme and the protein complex containing βTrCP, polyubiquitination of IκBα peptide was dependent on the presence of Cdc34, while Ubc5 only stimulated mono‐ and di‐ubiquitination.
Establishing a cost recovery system for drugs, Bo, Sierra Leone
Authors: Thuray H , Samai O , Fofana P , Sengeh P ,   (The Bo PMM Team)
Published: July 2002

Preliminary studies:Focus group discussions and health facility inventories in a rural district hospital indicated a shortage of basic drugs and supplies.Interventions:The project procured obstetric drugs and supplies through a commercial source outside normal government channels. They were managed through the standard district system and made available at prices calculated to cover costs and an 85% mark‐up. Obstetric emergency drug packs were created to ensure 24‐h availability. Patients from the study area unable to pay at time of treatment were given credit and followed up.Results:No shortages of obstetric drugs occurred after interventions were introduced. Prices of drugs to obstetric patients ranged from 46% to 68% of the prices charged by the government hospital and private pharmacies. Of 26 obstetric patients from the study area who received drugs in 1993, 12 paid in full and nine paid in part. The total amount billed during 1993 was US $2537, of which $1451 was recovered.Costs:Improvements cost approximately $17 500, of which 83% was used for the initial supply of drugs.Conclusions:Cost recovery systems for drugs can help maintain consistent drug supplies at health facilities. Careful pricing is critical. For the system to be sustainable, the mark‐up must cover defaulters. © 1997 International Federation of Gynecology and Obstetrics
Evidence‐based intrapartum care: evidence from the Cochrane library
Authors: Neilson J.P.
Published: July 2002

Objective: To describe the concepts on which systematic reviews of randomized controlled trials are based, and to explore the evidence for effectiveness of clinical interventions during labor and delivery. Methods: Review of systematic reviews of interventions during labor and delivery, published in the Cochrane Library. Results: Some commonly applied clinical interventions are supported by evidence; others are not. Conclusion: It is possible to classify interventions during labor and delivery according to their effectiveness, to improve clinical care, identify priorities where resources are scarce, and help set clinical research agendas.
Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor‐β1 in monolayer and insulin‐like growth factor‐I in a three‐dimensional matrix
Authors: Worster Allison A. , Brower‐Toland Brent D. , Fortier Lisa A. , Bent Stephen J. , Williams Janice , Nixon Alan J.
Published: July 2002

This study evaluated chondrogenesis of mesenchymal progenitor stem cells (MSCs) cultured initially under pre‐confluent monolayer conditions exposed to transforming growth factor‐β (TGF‐β), and subsequently in three‐dimensional cultures containing insulin‐like growth factor I (IGF‐I). Bone marrow aspirates and chondrocytes were obtained from horses and cultured in monolayer with 0 or 5 ng of TGF‐β1 per ml of medium for 6 days. TGF‐β1 treated and untreated cultures were distributed to three‐dimensional fibrin disks containing 0 or 100 ng of IGF‐I per ml of medium to establish four treatment groups. After 13 days, cultures were assessed by toluidine blue staining, collagen types I and II in situ hybridization and immunohistochemistry, proteoglycan production by [Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor‐β1 in monolayer and insulin‐like growth factor‐I in a three‐dimensional matrix35S]‐sulfate incorporation, and disk DNA content by fluorometry. Mesenchymal cells in monolayer cultures treated with TGF‐β1 actively proliferated for the first 4 days, developed cellular rounding, and formed cell clusters. Treated MSC cultures had a two‐fold increase in medium proteoglycan content. Pretreatment of MSCs with TGF‐β1 followed by exposure of cells to IGF‐I in three‐dimensional culture significantly increased the formation of markers of chondrocytic function including disk proteoglycan content and procollagen type II mRNA production. However, proteoglycan and procollagen type II production by MSC's remained lower than parallel chondrocyte cultures. MSC pretreatment with TGF‐β1 without sequential IGF‐I was less effective in initiating expression of markers of chondrogenesis. This study indicates that although MSC differentiation was less than complete when compared to mature chondrocytes, chondrogenesis was observed in IGF‐I supplemented cultures, particularly when used in concert with TGF‐β1 pretreatment. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Expression ofneu and Neu differentiation factor in the olfactory mucosa of rat
Authors: Salehi‐Ashtiani Kourosh , Farbman Albert I.
Published: July 2002

The growth and differentiation of olfactory sensory neurons are regulated tightly. We had shown previously, by immunohistochemistry, that transforming growth factor‐α (TGF‐α) and epidermal growth factor (EGF) receptor are present in the olfactory epithelium of untreated adult rats and that TGF‐α is a potent mitogen of olfactory epitheliumin vitro. Expression of EGF receptor and TGF‐α was detected primarily in horizontal basal cells and supporting cells but rarely in globose basal cells, which suggested that EGF receptor is not a likely candidate for the mitotic regulator of sensory neurons. In order to expand the search for candidate regulators, we have now examined other members of the EGF family of receptors and ligands. By utilizing reverse transcriptase‐polymerase chain reaction (RT‐PCR) methodology, we have detected the messenger RNA encoding the protein of theneu gene (p185neu) and Neu differentiation factor (NDF) isoforms in the olfactory mucosa. Immunohistochemical localization of p185neu and NDF indicates expression of these proteins in the olfactory epithelium of adult rats in regions where globose basal cells and immature sensory neurons are found, as well as in the ensheathing cells of the olfactory nerve. The presence ofneu and NDF transcripts in the olfactory tissue and the localization of their encoded polypeptides to proliferative regions of the epithelium suggest involvement of these gene products in the regulated proliferation/differentiation of the sensory neurons.