bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of single‐chain Fv genes encoding antibodies specific for Drosophila Poxn protein
Authors: Hassanzadeh Gh Gholamreza , De Silva Kumudu S.K. , Dambly-Chaudière Christine , Brys Lea , Ghysen Alain , Hamers Raymond , Muyldermans Serge , De Baetselier Patrick
Published: July 2002

The usefulness of intrabodies as specific inhibitors of gene function has been extensively demonstrated in cell culture assays. However, very few experiments have been conducted with intrabodies expressed in whole organisms. To evaluate the intrabody technology in Drosophila, we focused on poxn protein, since its effects can be easily studied. We purified the recombinant poxn protein. We next isolated three single‐chain variable fragments (scFv) which specifically recognize poxn protein. Two scFvs, designated α‐Poxn2 and α‐Poxn4, react with both denatured and native Poxn with half maximal inhibition values of 100 nM and 40 nM, respectively. The α‐Poxn5 scFv also recognizes denatured Poxn but either does not recognize native Poxn or its half maximal inhibition value for native Poxn is high.
Members of the fatty acid‐binding protein family inhibit cell‐free protein synthesis
Authors: Zimmerman Aukje W , Veerkamp Jacques H
Published: July 2002

Fatty acid‐binding proteins (FABPs) are 15‐kDa cytosolic proteins which are involved in the intracellular binding and targeting of fatty acids. Some members have been implicated in the regulation of cell growth and differentiation. In this study we investigated the effect of a series of FABPs and heart FABP (H‐FABP) mutants on cell‐free protein synthesis. Human myelin, intestinal, heart and brain FABP showed a dose‐dependent inhibition of in vitro mRNA translation. Adipocyte, liver and epidermal types had no effect. The inhibition was not influenced by delipidation and for H‐FABP mutants not related to their affinity for fatty acids. Our results indicate that some FABPs may modulate cell growth and/or differentiation by inhibition of protein synthesis.
Autocatalytic activation of human legumain at aspartic acid residues
Authors: Halfon Sherin , Patel Sejal , Vega Felix , Zurawski Sandra , Zurawski Gerard
Published: July 2002

Human legumain was characterized following overexpression in a murine cell line as the C‐terminal Ig‐fusion protein. Upon acid treatment, the prolegumain autoproteolyzed distal to two aspartic acid residues to yield a highly active form. The ability of mature legumain to cleave after aspartic acid residues was confirmed with a small peptide substrate. Substitution of alanine for the putative catalytic cysteine, or for either of two strictly conserved histidine residues, partly or wholly eliminated autoactivation but not the ability of wild‐type legumain to correctly process the variants to the properly sized proteins.
Longevity of lobsters is linked to ubiquitous telomerase expression
Authors: Klapper Wolfram , Kühne Karen , Singh Kumud K , Heidorn Klaus , Parwaresch Reza , Krupp Guido
Published: July 2002

Mammals have high growth rates in embryonic and juvenile phases and no growth in adult and senescent phases. We analyzed telomerase activity in a fundamentally different animal which grows indeterminately. Lobsters (Homarus americanus) grow throughout their life and the occurrence of senescence is slow. A modified TRAP assay was developed and the lobster telomeric repeat sequence TTAGG was determined. We detected telomerase activities which were dependent on RNA and protein components, required dGTP, dATP and dTTP, but not dCTP. Telomerase products with a five nucleotide periodicity were generated. High telomerase activities were detected in all lobster organs. We conclude that telomerase activation is a conserved mechanism for maintaining long‐term cell proliferation capacity and preventing senescence, not only in cellular models or embryonic life stages but also in adult multicellular organisms.
Aah VI, a novel, N‐glycosylated anti‐insect toxin from Androctonus australis hector scorpion venom: isolation, characterisation, and glycan structure determination
Authors: Hassani O , Loew D , Van Dorsselaer A , Papandréou M.J , Sorokine O , Rochat H , Sampieri F , Mansuelle P
Published: July 2002

Aah VI was isolated from the venom of the North African scorpion, Androctonus australis hector. It is the first glycosylated neurotoxin from scorpion venom to be described. It was not toxic to mice, when injected intracerebroventricularly at a dose of 1.2 μg per animal. However, it had typical activity in Blatella germanica cockroaches resulting in gradual paralysis and very low toxicity (LD50=8.5 μg/g of animal). It consists of 66 amino acid residues and is heterogeneously N‐glycosylated at a single site, on asparagine 9, of the Asn‐Gly‐Thr sequence. The potential N‐glycosylation site was deduced from automatic Edman degradation and amino acid analysis, and glycan heterogeneity was evidenced by ESMS. Determination of the N‐glycan structures (dHex, Hex and HexNAc) was assessed by nanoESMS/MS with picomolar amounts of sample. Current knowledge of N‐glycan structure and composition suggests that the glycan structures are derived from a common core.
Cloning of a novel four repeat protein related to voltage‐gated sodium and calcium channels
Authors: Lee Jung-Ha , Cribbs Leanne L. , Perez-Reyes Edward
Published: July 2002

Cloning has led to the discovery of more ion channels than predicted by functional studies, yet there remain channels that have not been cloned. We report the cloning of a novel protein that contains the four domain structure found in voltage‐gated Ca2+ and Na+ channels. Phylogenetic relationships suggested that the protein might have diverged from an ancestral four repeat channel before the divergence of Ca2+ and Na+ channels. Northern blot analysis showed that mRNA transcripts encoding the protein are expressed predominantly in the brain, moderately in the heart, and weakly in the pancreas. Despite extensive expression attempts, currents from the putative channel were not detected. Based on its sequence, we propose that the novel protein might be a voltage‐activated cation channel with unique gating properties.
Distribution of exogenous 25‐hydroxycholesterol in Hep G2 cells between two different pools
Authors: Kisseleva Anastassia F. , Goryunova Ludmila E. , Medvedeva Natalia V. , Alquier Christian , Morozkin Andrei D. , Misharin Alexander Yu.
Published: July 2002

Binding of [26,27‐3H]25‐hydroxycholesterol (25HC) to human hepatoma Hep G2 cells was saturated within 120 min. Two intracellular pools of 25HC were identified in a pulse‐chase experiment: (i) an exchangeable pool which was in dynamic equilibrium with 25HC in the medium (t
1/2 of reversible exchange 15 min) and (ii) an unexchangeable pool which remained in cells during incubation in medium containing LPDS. 25HC from the exchangeable pool inhibits cholesterol biosynthesis, decreases the HMG CoA reductase mRNA level and stimulates cholesterol acylation. 25HC from the unexchangeable pool was partially bound to cytosolic proteins and apparently utilized for metabolic transformation. Incubation of Hep G2 cells with [26,27‐3H]25HC in the presence of a 30‐fold molar excess of 3β‐hydroxy‐5α‐cholest‐8(14)‐en‐15‐one was found to cause (i) 2‐fold decrease in the binding of [26,27‐3H]25HC to cytosolic proteins (sedimentation constant of radioactive complex was 4–5 S) and (ii) the 35% inhibition of 25HC transformation to polar metabolites.
Complement component C8γ is expressed in human fetal and adult kidney independent of C8α
Authors: Trojer Patrick , Wojnar Petra , Merschak Petra , Redl Bernhard
Published: July 2002

Human complement component C8γ is an unusual complement factor since it shows no homology to other complement proteins but is a member of the lipocalin superfamily. So far, it has been found exclusively in plasma, covalently linked to C8α by disulfide bridging. We have used dot blot and Northern blot analyses of a large number of different human tissues to survey systematically the expression pattern of C8γ. Our experiments clearly showed that besides in liver, this gene is also expressed in fetal and adult kidney. Renal expression of C8γ is not dependent on C8α expression, since we could not detect C8α expression in kidney. Thus its physiological function is not restricted to a specific action in association with complement components. As a prerequisite for further characterization of the structure and binding activities of the uncomplexed C8γ, we have expressed the encoding cDNA in Escherichia coli. To increase the probability for proper folding of the characteristic intramolecular disulfide bridge the recombinant protein was produced by secretion to the periplasm.
Negative cooperativity between juxtaposed E‐box and cAMP/TPA responsive elements in the cholecystokinin gene promoter
Authors: Rourke Ian J , Hansen Thomas vO , Nerlov Claus , Rehfeld Jens F , Nielsen Finn C
Published: July 2002

The promoter of the cholecystokinin (CCK) gene possesses evolutionary conserved juxtaposed E‐box and cAMP/TPA responsive elements (CRE/TRE). We have examined the functional interaction of these two sites. As previously noted, c‐Jun/c‐Fos heterodimers greatly increase promoter activity through association with the CRE/TRE. Mutation of the E‐box enhanced the activation by c‐Jun/c‐Fos, as well as stimulation by forskolin and bFGF, that acts through the CRE/TRE site. Moreover, c‐Jun/c‐Fos stimulation was inhibited by co‐expression of c‐Myc and Max. The results indicate that factors associating with the E‐box exhibit a negative cooperative effect on the activation via the CRE/TRE element. We propose that this mechanism plays a significant role in CCK gene transcription and other genes with juxtaposed E‐box and CRE/TRE.
Isolation and characterisation of oxygen evolving thylakoids from the marine prokaryote Prochloron didemni
Authors: Christen G. , Stevens G. , Lukins P.B. , Renger G. , Larkum A.W.D.
Published: July 2002

The present study describes the first successful attempt to isolate oxygen evolving thylakoids and thylakoid fragments from the marine prokaryote Prochloron didemni, a member of the recently discovered group of prochlorophytes. Oxygen evolving thylakoid membranes and fragments were isolated from seawater suspended cells of Prochloron didemni by passage of the cells through a Yeda press and subsequent differential centrifugation of the broken material. Three fractions were collected at 1000×g, 5000×g, and 30 000×g and identified by light microscopy as cells (and their fragments), thylakoids and membrane fragments, respectively. Pigment content, oxygen evolution rate and 77 K fluorescence spectra of these fractions were virtually identical. This finding indicates that the membrane fragments obtained are not enriched in photosystem II. The P680+
reduction kinetics of thylakoid membrane fragments were determined by monitoring flash induced absorption changes at 830 nm and analysing the time course of their decay. The multiphasic relaxation kinetics and their modification by NH2OH were found to be similar to those observed in cyanobacteria and plants. These findings provide an independent line of evidence for the idea of a high conservation of the basic structural and functional pattern of the water oxidising complex in all organisms that perform oxygenic photosynthesis.
Inorganic Fe2+ formation upon Fe‐S protein thermodestruction in the membranes of thermophilic cyanobacteria: Mössbauer spectroscopy study
Authors: Kaurov Yu.N. , Novakova A.A. , Davletshina L.N. , Aleksandrov A.Yu. , Khval'kovskaya E.A. , Semin B.K. , Belevich N.P. , Ivanov I.I. , Rubin A.B.
Published: July 2002

A model description of the Mössbauer spectrum (80 K) of native membranes of the thermophilic cyanobacterium Synechococcus elongatus is suggested on the basis of the known values of quadrupole splitting (ΔE
Q) and isomer shift (δ
Fe) for the iron‐containing components of the photosynthetic apparatus. Using this approach, we found that heating the membranes at 70–80 K results in a decrease of doublet amplitudes belonging to FX, FA, FB and ferredoxin and simultaneous formation of a new doublet with ΔE
Q=3.10 mm/s and δ
Fe=1.28 mm/s, typical of inorganic hydrated forms of Fe2+. The inhibition of electron transfer via photosystem I to oxygen, catalyzed by ferredoxin, occurs within the same range of temperatures. The data demonstrate that the processes of thermoinduced Fe2+ formation and distortions in the photosystem I electron transport in the membranes are interrelated and caused mainly by the degradation of ferredoxin. The possible role of Fe2+ formation in the damage of the photosynthetic apparatus resulting from heating and the action of other extreme factors is discussed.
Reconstruction of the dihydropyridine site in a non‐L‐type calcium channel: the role of the IS6 segment
Authors: Lacinová Lubica , Klugbauer Norbert , Hu Min , Hofmann Franz
Published: July 2002

Mutations of eight to nine amino acids of IIIS5, IIIS6 and IVS6 segments were shown to reconstruct the dihydropyridine (DHP) interaction site in the non‐L‐type α1E or α1A calcium channels. The reconstructed site enabled enantiomer‐selective inhibition and activation of the expressed chimeras by DHPs but failed to transfer voltage dependence of the current inhibition. Here we show that transfer of four non‐conserved amino acids from the IS6 segment to the DHP‐sensitive α1E chimera increased the inhibition by (+)isradipine at the hyperpolarized membrane potential of −100 mV and enhanced the voltage‐dependent block.
CA2+‐dependent interaction of the trpl cation channel and calmodulin
Authors: Trost Claudia , Marquart Andrea , Zimmer Stephanie , Philipp Stephan , Cavalié Adolfo , Flockerzi Veit
Published: July 2002

The transient receptor potential‐like ion channel from Drosophila melanogaster was originally identified as a calmodulin binding protein (Philips et al., 1992) involved in the dipterian phototransduction process. We used a series of fusion proteins and an epitope expression library of transient receptor potential‐like fusion proteins to characterize calmodulin binding regions in the transient receptor potential‐like channel through the use of [125I]calmodulin and biotinylated calmodulin and identified two distinct sites at the C‐terminus of the transient receptor potential‐like ion channel. Calmodulin binding site 1, predicted from searching of the primary structure for amphiphilic helices (Philips et al., 1992), covers a 16 amino acid sequence (S710–I725) and could only be detected through biotinylated calmodulin. Calmodulin binding site 2 comprises at least 13 amino acids (K859ETAKERFQRVAR871) and binds both [125I]calmodulin and biotinylated calmodulin. Both sites (i) bind calmodulin at least in a one to one stoichiometry, (ii) differ in their affinity for calmodulin revealing apparent K
i values of 12.3 nM (calmodulin binding site 1) and 1.7 nM (calmodulin binding site 2), respectively, (iii) bind calmodulin only in the presence of Ca2+ with 50% of site 1 and site 2, respectively, occupied by calmodulin in the presence of 0.1 μM (calmodulin binding site 1) and 3.3 μM Ca2+ (calmodulin binding site 2) and give evidence that (iv) a Ca2+‐calmodulin‐dependent mechanism contributes to transient receptor potential‐like cation channel modulation when expressed in CHO cells.
Specific binding of glucosaminylmuramyl peptides to histones
Authors: Golovina Tatyana , Fattakhova Gulnara , Swiderek Kristine , Makarov Eugeni , Bovin Nikolai , Shively John , Nesmeyanov Vladimir
Published: July 2002

Intracellular N‐acetylglucosaminylmuramyl peptide‐binding proteins of murine macrophages and myelomonocytic WEHI‐3 cells were characterized. SDS‐PAGE and Western blotting revealed proteins with molecular masses of 18, 32 and 34 kDa retaining the ability to specifically bind glucosaminylmuramyl dipeptide. The inhibition analysis demonstrated that only biologically active muramyl peptides but not inactive analogs or fragments of glucosaminylmuramyl dipeptide could inhibit glucosaminylmuramyl dipeptide‐binding to these proteins. Purification of these proteins and sequencing of peptides obtained after in‐gel trypsin digestion enabled us to identify the above mentioned proteins as histones H1 and H3. These findings suggest that nuclear histones might be target molecules for muramyl peptides.
Analysis of the in vivo interaction between a basic repressor and an acidic activator
Authors: Wellhausen Anne , Lehming Norbert
Published: July 2002

The artificial basic repressor SSB24 represses transcription of a reporter construct activated by GCN4. We show that the positively charged SSB24 and the negatively charged acidic activator GCN4 interact in vitro and in vivo. However, deleting the interaction domain from the GCN4 activator does not result in loss of repression by SSB24. Similarly, transcription activated by the holoenzyme component SRB2 is repressed, although SSB24 and SRB2 do not interact. Repression by SSB24 therefore does not depend on the observed protein‐protein interaction between SSB24 and GCN4.
Heparin binding to cobra basic phospholipase A2 depends on heparin chain length and amino acid specificity
Authors: Lin Yi-Hung , Lee Shao-Chen , Chang Payne Y. , Rajan P.K. , Sue Shih-Che , Wu Wen-guey
Published: July 2002

Heparin is shown to bind specifically to the carboxy‐terminal region of toxic type I phospholipase A2 from Naja nigricollis (N‐PLA2) by competition assay using synthetic polypeptides and heparin affinity chromatography. The binding strength is seen to depend on heparin chain length and the presence of N‐sulfate groups of heparin. It is observed that both electrostatic and non‐electrostatic interactions are involved in the specific binding of heparin to the carboxy‐terminus. When heparin's size is at least a decasaccharide, about two molecules of N‐PLA2 bind to one molecule of heparin, as evidenced by the chemical estimate of protein to carbohydrate ratio in such N‐PLA2/heparin complexes. Based on such a stoichiometric measurement and computer modeling of the N‐PLA2/heparin complex, it is suggested that the binding sites of the two N‐PLA2 molecules on one heparin molecule lie on the opposite sides of the heparin chain.
PKC‐dependent phosphorylation of the p97 repressor regulates the transcription of aldolase A L‐type promoter
Authors: Costanzo Paola , Lupo Angelo , Medugno Lina , D'Agostino Paola , Zevino Chiara , Izzo Paola
Published: July 2002

Expression of mouse aldolase A L‐type mRNA is negatively modulated by a cis element (AldA‐NRE), located within the aldolase A distal promoter (pL). AldA‐NRE interacts with a 97‐kDa repressor protein (p97), which binds DNA in a cell cycle‐dependent manner. We demonstrate that the binding between AldA‐NRE and p97 decreases during differentiation of human Caco‐2 cells and is inversely correlated with L‐type mRNA expression. Phosphorylation of the p97 repressor weakened its DNA binding activity in differentiated Caco‐2 cells, while dephosphorylation enhanced the binding in proliferating cells. Stimulation of protein kinase C (PKC) in vivo decreased the binding of p97 to AldA‐NRE and stimulated transcription, while inhibition of PKC stimulated p97 binding and downregulated transcription. These findings suggest that PKC is a mediator of the binding and silencing function of the p97/AldA‐NRE repressor complex.
Interaction with free β′ subunit unmasks DNA‐binding domain of RNA polymerase σ subunit
Authors: Kulbachinskiy Andrey , Mustaev Arkady , Goldfarb Alex , Nikiforov Vadim
Published: July 2002

The promoter recognition site on the σ70 initiation factor is shielded from interaction with DNA unless σ70 is bound to the core component of RNA polymerase (RNAP). It is shown that interaction of σ70 with the isolated β′ subunit of Escherichia coli RNAP is sufficient to induce unshielding of the DNA binding site. Using UV‐induced DNA‐protein cross‐linking we demonstrate that free β′ stimulates specific cross‐links between region 2 of the σ70 polypeptide and a fragment of the non‐template promoter strand containing the TATAAT sequence. Thus the σβ′ subassembly of RNAP can assume a functionally competent conformation independently of the bulk of the RNAP core.
Arsenic trioxide induces apoptosis in neuroblastoma cell lines through the activation of caspase 3 in vitro
Authors: Akao Yukihiro , Nakagawa Yoshihito , Akiyama Kiyotaka
Published: July 2002

Arsenic trioxide (As2O3) induces clinical remission in acute promyelocytic leukemia, even in all‐trans retinoic acid‐refractory cases, with minimal toxicity at low (1–2 μM) concentration. We exposed various neuroblastoma cell lines to As2O3 at a concentration of 2 μM: as a result, seven of 10 neuroblastoma cell lines underwent apoptosis characterized by morphological changes and nucleosomal DNA fragmentation. As2O3‐induced apoptosis in neuroblastoma cells was shown to occur through the activation of caspase 3, as judged from Western blot analysis and apoptosis inhibition assay. It seemed that the sensitivity of neuroblastoma cells to As2O3 was inversely proportional to their intracellular level of reduced glutathione. Taken together these results indicate that As2O3 would be a candidate as a therapeutic agent for treatment of neuroblastoma, which is a solid tumor, not only by systemic therapy but also by local therapy.
Translocon‐associated protein α transcripts are induced by granulocyte‐macrophage colony‐stimulating factor and exhibit complex alternative polyadenylation
Authors: Hirama Toshiyasu , Miller Carl W. , Koeffler H.Phillip
Published: July 2002

The cloning of full length cDNA for the translocon‐associated protein α subunit, previously called signal sequence receptor α, is reported as a result of differential display experiments in search of genes induced by granulocyte‐macrophage colony‐stimulating factor. Its messenger RNA was more abundant in growing cells than in either factor‐deprived cells or quiescent cells and comprised four species, each having microheterogeneity, as a result of complex alternative polyadenylation apparently dependent on arrays of non‐canonical polyadenylation signals. Radiation hybrid mapping of the gene showed that the gene is on the short arm of chromosome 6.
Subcellular distribution of farnesyl protein transferase in rat liver
Authors: Grünler Jacob , Parmryd Ingela
Published: July 2002

Farnesyl protein transferase (FPT) activity was measured in rat liver subcellular fractions by using an unspecific acceptor for the farnesyl groups. The highest specific activity was found in mitochondria and it exceeded that of the microsomes three‐fold. Considerably lower specific activities were found in the nuclei and cytosol. Further subfractionation revealed that the mitochondrial FPT activity is located in the matrix. The β‐subunit of the mitochondrial enzyme has an apparent molecular mass of 46 kDa, which is similar to its cytosolic counterpart. The results suggest that protein farnesylation can take place in a number of subcellular organelles.
The in vitro synthesized and processed human insulin receptor precursor binds insulin
Authors: Tsuda Shawn , Kaihara Masahiro , Zhou Xiangjun , Britos Dean , Arakaki Richard
Published: July 2002

The cell‐free examination of the human insulin receptor during biogenesis may provide a greater understanding of the elements that contribute to the acquisition of receptor function. The insulin receptor precursor components were produced in a cell‐free system and the insulin binding ability of the [35S]methionine‐labeled translation products was determined. The processed proreceptor represented by a 190 kDa band was retained on insulin‐linked biotin‐streptavidin agarose or an insulin column. The insulin binding 190 kDa band migrated slower than the non‐binding 190 kDa band on SDS‐PAGE which suggests that covalent modifications account for these differences. The trypsin‐digested product of the 190 kDa proreceptor was also retained on insulin‐linked biotin‐streptavidin agarose, however the α‐subunit precursor was retained on insulin agarose to a much lesser degree. We conclude that a significant fraction of the processed, in vitro translated insulin proreceptor acquires insulin binding ability.
Bromine K‐edge EXAFS studies of bromide binding to bromoperoxidase from Ascophyllum nodosum
Authors: Dau Holger , Dittmer Jens , Epple Matthias , Hanss Jan , Kiss Erzsébet , Rehder Dieter , Schulzke Carola , Vilter Hans
Published: July 2002

Bromine K‐edge EXAFS studies have been carried out for bromide/peroxidase samples in Tris buffer at pH 8. The results are compared with those of aqueous (Tris‐buffered) bromide and vanadium model compounds containing Br‐V, Br‐C(aliphatic) and Br‐C(aromatic) bonds. It is found that bromide does not coordinate to the vanadium centre. Rather, bromine binds covalently to carbon. A possible candidate is active site serine.
Different cation binding to the I domains of α1 and α2 integrins: implication of the binding site structure
Authors: Obšil Tomáš , Hofbauerová Kateřina , Amler Evžen , Teisinger Jan
Published: July 2002

In the present work, we studied the interactions of recombinant α1 and α2 integrin I domains with cations Tb3+, Mn2+, Mg2+ and Ca2+. We observed that α1 and α2 I domains bind these cations with significantly different characteristics. The binding of Mg2+ by the α1 I domain was accompanied by significant changes of tryptophan fluorescence which could be interpreted as a conformational change. Comparison of the α1 integrin I domain structure obtained by comparative modeling with a known structure of the α2 integrin I domain shows distinct differences in the metal ion binding sites which could explain the differences in cation binding.
Alternative exon 3 splicing of the human major protein zero gene in white blood cells and peripheral nerve tissue
Authors: Besançon R. , Prost A.L. , Konecny L. , Latour P. , Petiot P. , Boutrand L. , Kopp N. , Mularoni A. , Chamba G. , Vandenberghe A.
Published: July 2002

The major protein zero (MPZ) is involved in peripheral myelin folding. Using nested reverse transcription‐PCR, we amplified several fragments of MPZ mRNAs in white blood cells and in peripheral nerve tissue. Cloning of PCR products revealed the existence of three alternative splicing patterns: one resulted in the complete loss of exon 3 and two others induced partial skipping of the exon 3 sequence. All three alternative splicing mechanisms produced a frame‐shift and created an identical premature stop codon in exon 4. We conclude that the existence of these MPZ RNA transcript variants may be the result of deliberate splicing decisions and may have functional implications in the cell.
Δ9‐Tetrahydrocannabinol induces apoptosis in human prostate PC‐3 cells via a receptor‐independent mechanism
Authors: Ruiz Lidia , Miguel Alberto , Dı́az-Laviada Inés
Published: July 2002

The effect of Δ9‐tetrahydrocannabinol (THC), the major psycho‐active component of marijuana, in human prostate cancer cells PC‐3 was investigated. THC caused apoptosis in a dose‐dependent manner. Morphological and biochemical changes induced by THC in prostate PC‐3 cells shared the characteristics of an apoptotic phenomenon. First, loss of plasma membrane asymmetry determined by fluorescent anexin V binding. Second, presence of apoptotic bodies and nuclear fragmentation observed by DNA staining with 4′,6‐diamino‐2‐phenylindole (DAPI). Third, presence of typical ‘ladder‐patterned’ DNA fragmentation. Central cannabinoid receptor expression was observed in PC‐3 cells by immunofluorescence studies. However, several results indicated that the apoptotic effect was cannabinoid receptor‐independent, such as lack of an effect of the potent cannabinoid agonist WIN 55,212‐2, inability of cannabinoid antagonist AM 251 to prevent cellular death caused by THC and absence of an effect of pertussis toxin pre‐treatment.
Identification of a novel 300‐kDa factor termed IκBαE3‐F1 that is required for ubiquitinylation of IκBα
Authors: Suzuki Hiroshi , Kobayashi Masato , Takeuchi Masahiro , Furuichi Kiyoshi , Chiba Tomoki , Tanaka Keiji
Published: July 2002

Destruction of IκB by ubiquitinylation is required for signal‐dependent activation of NF‐κB. The IκBα ubiquitin‐ligase activity associated with phosphorylated IκBα (pIκBα) in HeLa cells was almost completely lost by washing under stringent conditions including 1 M NaCl; nevertheless, an SCFβTrCP complex containing Skp1, Cullin‐1, and F‐box/WD40 protein βTrCP was still bound to pIκBα, suggesting the existence of a putative factor that is loosely associated with pIκBα and may collaborate with SCFβTrCP. The factor was named IκBαE3‐F1 and was partially purified from HeLa cells. Gel filtration analysis revealed that IκBαE3‐F1 has an apparent molecular mass of approximately 300 kDa.
A novel calcitonin receptor gene in human osteoclasts from normal bone marrow
Authors: Nishikawa Takuji , Ishikawa Hirokazu , Yamamoto Seizo , Koshihara Yasuko
Published: July 2002

The calcitonin receptor (CTR) gene in human osteoclasts formed in a human bone marrow cell culture system was examined by reverse transcription‐polymerase chain reaction (RT‐PCR). The RT‐PCR results indicated that the 5′‐untranslated region (5′UTR) was different between CTR mRNAs in human osteoclasts and in a mammary tumor cell line, MCF‐7 cells. We isolated the 5′UTR of the CTR gene from human osteoclasts, whose sequence had only 28.6% identity with that of other CTR genes reported until now. In a radioligand binding assay, COS‐1 cells transfected with the osteoclast CTR gene bound to [125I]human CT (hCT). These results provided evidence that the CTR gene cloned from human osteoclasts was expressed functionally and its coding protein was identical to MCF‐7 cell CTR.
SCOP, a novel gene product expressed in a circadian manner in rat suprachiasmatic nucleus
Authors: Shimizu Kimiko , Okada Masato , Takano Atsuko , Nagai Katsuya
Published: July 2002

To elucidate the mechanism of the circadian rhythm, genes differentially expressed during subjective day and night in the rat suprachiasmatic nucleus (SCN), a circadian oscillator in mammals, were surveyed by a differential display method. We isolated a novel gene, scop (CN ircadian scillatory rotein), that was expressed in a circadian manner in the SCN. SCOP protein is predominantly expressed in the brain and has domains including a pleckstrin homology domain, leucine‐rich repeats, a protein phosphatase 2C‐like domain and a glutamine‐rich region. The structural feature of SCOP protein suggests its role in the intracellular signaling in the SCN.
Cloning of mouse mitochondrial thymidine kinase 2 cDNA
Authors: Wettin Katarina , Johansson Magnus , Zheng Xinyu , Zhu Chaoyong , Karlsson Anna
Published: July 2002

Phosphorylation of anti‐viral nucleoside analogs by mitochondrial thymidine kinase 2 (TK2) has been implicated as a mechanism for the mitochondrial toxicity caused by several of these compounds. We have cloned the cDNA of mouse TK2 and shown that the enzyme is targeted to the mitochondria when expressed in fusion with the green fluorescent protein. This is the first report on the cloning of a mitochondrial TK2 and will contribute to elucidate the role of TK2 in the pharmacological activation of nucleoside analogs.
The aspartic proteinase from the rodent parasite Plasmodium berghei as a potential model for plasmepsins from the human malaria parasite, Plasmodium falciparum
Authors: Humphreys Michelle J. , Moon Richard P. , Klinder Annette , Fowler Sylvia D. , Rupp Katharina , Bur Daniel , Ridley Robert G. , Berry Colin
Published: July 2002

The gene encoding an aspartic proteinase precursor (proplasmepsin) from the rodent malaria parasite Plasmodium berghei has been cloned. Recombinant P. berghei plasmepsin hydrolysed a synthetic peptide substrate and this cleavage was prevented by the general aspartic proteinase inhibitor, isovaleryl pepstatin and by Ro40‐4388, a lead compound for the inhibition of plasmepsins from the human malaria parasite Plasmodium falciparum. Southern blotting detected only one proplasmepsin gene in P. berghei. Two plasmepsins have previously been reported in P. falciparum. Here, we describe two further proplasmepsin genes from this species. The suitability of P. berghei as a model for the in vivo evaluation of plasmepsin inhibitors is discussed.
Phosphorylation of the Spi‐B transcription factor reduces its intrinsic stability
Authors: Ray-Gallet Dominique , Moreau-Gachelin Françoise
Published: July 2002

The Spi‐B transcription factor is an Ets protein expressed in B lymphoid cells and closely related to the Spi‐1/PU.1 oncoprotein. By mutational analysis, we showed that Spi‐B is phosphorylated by casein kinase II in vitro on four serine residues. Mutation of these four serines to alanines prevented the phosphorylation of Spi‐B in vivo, increased the ability of Spi‐B to transactivate expression of a reporter gene and led to a decrease of Spi‐B stability. We propose that the phosphorylation of Spi‐B may participate in the modulation of Spi‐B functional activity by controlling its intracellular protein level.
Assessment of gestational age based on ultrasonic femur length after the first trimester: a simple mathematical correlation between gestational age (GA) and femur length (FL)
Authors: Honarvar M. , Allahyari M. , Dehbashi S.
Published: July 2002

Objective: The ultrasonic measurement of the fetal femur length is a sensitive and precise variable for estimation of fetal growth and development. The objective of this study was to predict gestational age in fetuses more than 13 weeks of gestation by ultrasound measurement of the femur length. Method: In this study, pregnant mothers were identified by the criteria of normalities such as: well‐known LMP, regular menstrual cycles, no use of OCP for the last 3 months, no smoking, no history of diabetes, etc. Result: The relation between gestational age and fetal femur length has been determined by cross‐sectional analysis of 900 normal fetuses (≥14 weeks of gestation) using real time ultrasonography. Mathematical modeling of the data has demonstrated that the femur growth curve is non‐linear beyond 13 weeks of gestation. With the aid of a scientific calculator the data were analyzed and a simple second grade equation has been derived: GA (weeks)=0.2622 FL (cm)+2 FL+11.5, S.D.∼±5 days (Honarvar's Formula 3). With the use of this data, the error in the estimation of GA given as FL is ±5days. Conclusion: This simple, new and accurate equation appears to be clinically reliable and easy to use and suggests that previous normal ultrasonic fetal femur length curves for another population may under‐ or overestimate normal fetal age for the Iranian population. Thus, our formula is an excellent means of estimating true gestational age.
Fetal fibronectin as a predictor of preterm delivery in twin gestations
Authors: Oliveira T , de Souza E , Mariani‐Neto C , Camano L
Published: July 2002

Objective: To evaluate fetal fibronectin as a predictor of premature delivery in twin pregnancies. Method: Cervicovaginal secretions were obtained from 52 pregnant women with twin pregnancies between 24 and 34 weeks of gestation. The secretions were analyzed to detect the presence of fetal fibronectin by immediate‐reading membrane test. The correlation between the presence of fetal fibronectin and preterm birth was evaluated. In addition, cervical dilatation and effacement were evaluated with each sampling. Result: The sensitivity, specificity, positive predictive value and negative predictive value to predict preterm delivery were 89.3, 50.0, 67.6, and 80.0%, respectively. A positive fetal fibronectin result was associated with a relative risk (RR) for preterm birth of 3.4 (95% CI, 1.2–9.5). A positive fetal fibronectin test associated with cervical dilatation or effacement increased the RR for preterm birth to 4.3 and 7.7, respectively, when compared with those with negative test and without cervical dilatation and effacement. Conclusion: Fetal fibronectin in the cervicovaginal secretions of patients with twin pregnancies is a sensitive predictor of preterm delivery. However, because of its low specificity, the fetal fibronectin test should be evaluated along with cervical changes for better identification of twins likely to develop preterm labor.
Fetal middle‐cerebral and umbilical artery flow assessments after glucose challenge test
Authors: Pardo J. , Orvieto R. , Rabinerson D. , Bar J. , Hod M. , Kaplan B.
Published: July 2002

Objective: To determine whether a change occurs in fetal middle‐cerebral and umbilical artery flow after glucose challenge testing. Method: Fetal middle‐cerebral and umbilical artery flow was assessed by Color Dopppler technology in 21 pregnant patients before and after the 50‐g glucose challenge test. The resistance index (IR) was evaluated separately for each vessel. Statistical significance was determined by Chi‐square test and repeated measurement analysis with two co‐variates. Results: After glucose ingestion the RI was significantly decreased in the middle‐cerebral artery, but not in the umbilical artery. A significant increase in RI was recorded in the umbilical artery when maternal plasma glucose level exceeded 102 mg%. Conclusion: Maternal ingestion of glucose significantly alters blood flow velocimetry in the fetal middle‐cerebral and umbilical arteries. These changes may lead to the misinterpretation of normal results. It is therefore recommended that sonographic flow velocimetry assessment not be performed early after concentrated glucose ingestion.
Cultured myf5 null and myoD null muscle precursor cells display distinct growth defects
Authors: Montarras Didier , Lindon Catherine , Pinset Christian , Domeyne Peggy
Published: July 2002

Summry— Myf‐5 and MyoD are the two muscle regulatory factors expressed from the myoblast stage to maintain the identity and to promote the subsequent differentiation of muscle precursor cells. To get insight into their role we have studied the capacity to proliferate and to differentiate of myf‐5 and myoD null myoblasts in primary cultures and in the subsequent passages. Our results indicate that myf‐5 null myoblasts differ from wild type (wt) myoblasts in that they undergo precocious differentiation: they become myogenin‐ and troponin T‐positive and fail to incorporate bromodeoxyuridine (BrdU) under culture conditions and at a time when wt cells are not yet differentiated and continue to proliferate. In primary cultures of myoD null cells, up to 60% of the cells were scored as myoblasts on the basis of the expression of myf‐5. These myoD‐deficient myoblasts, unlike myoD‐expressing cells, were poorly differentiating and displayed a severe growth defect that led to their elimination from the cultures: within a few passages myoblasts were absent from myoD‐deficient cultures, which mostly consisted of senescent cells. That a null mutation in either gene reduces the proliferative potential of cultured myoblasts raises the possibility that Myf‐5 and MyoD serve proliferation of muscle precursor cells.
Adenoviral‐mediated transfection of the lac‐z geneinto rat dissociated embryonic central nervous systemcells before and after seeding
Authors: Cot Christine , Privat Alain , Levallois Christine
Published: July 2002

The adenovirus carrying a reporter gene—the Lac‐Z gene—is well known toinfect central nervous system (CNS) cells in primary cell cultures. The percentage of infectedneurons with respect to the total number of neurons was studied in primary dissociated culturesas a function of the day of inoculation and the age of three rat CNS cultures : spinal cord,mesencephalon and cortex. Two methods of viral inoculation were compared : the firstinoculation was performed on the cultured cells at 2, 3 or 6 days in vitro (DIV) whereas the second inoculation was performed on the cell suspensions before seeding. All theinfected CNS cells had the same aspect as the control cultures. In the spinal cord and themesencephalic cultures, the glial cells were preferentially infected, especially when the cells wereinoculated at 6 DIV. In the cortical cultures, there were more infected neurons than infected glialcells. The number of CNS cells was lower when inoculation was performed at 6 DIV ascompared with 3 DIV. Very few infected GABA cells were found in the cultures. A highpercentage of infected neuronal cells relative to the total number of neuronal cells was foundwhen infection of the three types of cultures was performed on the dissociated embryonic cellsuspension before seeding.
Perception of species‐specific vocalizations in rats : role of the cholinergic septo‐hippocampal pathway and aging
Authors: Hörtnagl Heide , Groll‐knapp Elisabeth , Khanakah Gelas , Sperk Günther , Bubna‐littitz Hermann
Published: July 2002

The effect of a chemical lesion of the cholinergic septo‐hippocampal pathway induced by ethylcholine aziridinium (AF64A) on brain potentials evoked by species‐specific vocalization containing informations of high biological relevance was studied in young adult (10 months) and aged (24 months) rats by means of neocortical electroencephalographic recordings. In control rats, the perception of a rats vocalization in a life endangering situation (lasting 0.8 s) initiated an evoked potential followed by a late positive slow wave (LPSW)‐complex and a direct current shift with a duration of up to 16 s. Four months after treatment with AF64A (2 nmol into each of the lateral ventricles), the main negative component of the initial acoustic evoked potential (peak latency of about 60 ms after stimulus onset) was reduced (P = 0.04) both in young adult and aged rats. Further changes included a decrease of the late positive wave amplitude in young adult rats (P = 0.001) and a shorter duration of the LPSW‐complex in aged rats (P = 0.03). AF64A induced also changes in specific components revealed by Principle Component Analysis, but only in the group of young rats. A decrease in the slow wave component (factor 1, 3000–4000 ms after stimulus onset ; P = 0.02) was observed. Age per se affected the late positive potential shifts as indicated by a shorter latency of the late positive wave (P = 0.03).

A detailed analysis of the major neurotransmitter markers proved an almost exclusive reduction of the activity of choline acetyltransferase in the ventral and dorsal hippocampus (up to 60%), which was equal in young adult and aged rats. The irreversible loss in choline acetyltransferase activity, which was restricted to the hippocampus, was associated by a slight reduction in serotonergic function.

The present data suggest that the complex cognitive and emotional processes initiated by species‐specific vocalization appear to be affected by aging. Furthermore, as a consequence of a cholinergic deficit in the hippocampus, the integration processes essential for the recognition of the biological meaning of a species‐specific vocalization are considerably disturbed. These findings provide an experimental basis for studying disturbances in the perceptual response to stimuli of high emotional value in patients with hypocholinergic function as in Alzheimers disease.
Quality of life and treatment costs in schizophrenic outpatients, treated with depot neuroleptics
Authors: Dernovsek M.Z. , Prevolnik Rupel V. , Rebolj M. , Tavcar R.
Published: July 2002

Study aim was 1) to find out the influences on quality of life (QoL) of chronic outpatients with schizophrenia; 2) to calculate Quality Adjusted Life Years (QALY); and 3) to estimate direct 1-year treatment costs. In a 20% sample (100 males, 100 females) of schizophrenic outpatients from the Outpatients Clinic in Ljubljana, Slovenia receiving depot neuroleptics demographic, clinical, and treatment data were collected for the year 1996. The Krawiecka Scale, Global Assessment Scale (GAS), Abnormal Involuntary Movement Scale, Rating Scale for Drug-Induced Akathisia, Rating Scale for Extrapyramidal Side Effects, Quality of Life Scale (QLS), EQ-5D and QALY were used. Multivariate linear regression was used with the QLS score as dependent variable. The patients were on average 44 years old and had been treated for 14 years. The average GAS score was 70. GAS was positively related to the QLS score while the parkinsonism score was inversely correlated with QLS. The patients can expect to live for 10. 20 more QALY on average. The QoL on the EQ-5D scale was 0.73. The annual direct treatment costs amounted to $216,216 in 1996 prices. In well-adjusted chronic patients with schizophrenia the QoL seems to depend mostly on their psychosocial performance and side effects. Although rare, rehospitalisations accounted for one-half of all treatment expenses.
Barriers to the use of electroconvulsive therapy in the elderly: a European survey
Authors: Philpot M. , Treloar A. , Gormley N. , Gustafson L.
Published: July 2002

A postal survey was carried out to determine the clinical and legal guidelines governing the use of electroconvulsive therapy (ECT) in the countries of the wider Europe. Respondents from 23 of the 33 countries returned completed questionnaires. Considerable variation was found in the availability of ECT, the frequency of its use and associated legal procedures. However, there was a broad consensus with regard to the clinical indications. Access to the treatment was most frequently limited by financial or other resource constraints, political or legal restrictions.
Body experience in eating disorders before and after treatment: a follow-up study
Authors: Probst M. , Vandereycken W. , Van Coppenolle H. , Pieters G.
Published: July 2002

Since body dissatisfaction is an essential part of anorexia/bulimia nervosa, we wanted to assess the patient’s body experience before and after intensive treatment. Body experience was studied in 290 eating disorder patients, admitted to a specialised unit, after six months and again after one year using the Body Attitude Test (BAT), the Eating Disorder Inventory (EDI), and the Eating Disorder Evaluation Scale (EDES). The clearly negative body experience of eating disorder patients evolved in a positive way after therapy and this improvement lasted for up to one year after admission. EDI scores and Body Mass Index at the time of admission appeared to be the strongest predictors of the total BAT score at follow-up. Athough body dissatisfaction can be quite persistent in eating disorders, intensive treatment can substantially improve the patient’s body experience.
Autonomic dysfunction in patients with mild heart failure and coronary artery disease and the effects of add‐on β‐blockade
Authors: Tjeerdsma Geert , Szabó Balázs M. , van Wijk Leen M. , Brouwer Jan , Tio René A. , Crijns Harry J.G.M. , van Veldhuisen Dirk J.
Published: July 2002

Aim:

Autonomic impairment is related to the incidence of sudden death in chronic heart failure (CHF). Our objective was to study autonomic profiles in patients with mild CHF due to coronary artery disease, and to investigate the value of add‐on β‐blockade.

Methods and results:

Measures of autonomic function (plasma norepinephrine, heart rate [HR] variability, autonomic function testing), and exercise capacity, were compared between 24 patients with mild CHF, and 24 healthy controls. In this mechanistic study, we assessed the effect of 26 weeks metoprolol treatment in a double‐blind, randomized, placebo‐controlled design. All patients received metoprolol sustained release (200 mg; n = 12) or placebo (n = 12). Assessments were made at baseline and after 10 and 26 weeks' treatment. At baseline, norepinephrine levels were elevated, while HR variability parameters were decreased in patients vs. controls (both P < 0.05). Autonomic function testing showed only small differences, although significant alterations were observed with deep breathing and head up tilting (both P < 0.05). After 26 weeks', metoprolol did not affect exercise capacity or norepinephrine concentrations. In contrast, HR variability was markedly improved in metoprolol‐treated patients vs. placebo‐treated patients (P < 0.05). In particular, a shift toward normal in the sympathovagal balance was observed (P < 0.05). Autonomic function testing showed only small, and generally non‐significant trends after metoprolol.

Conclusions:

Marked autonomic abnormalities are already present in mild CHF, which may be (partially) reversed by metoprolol. These observations support the reported reduction of sudden death by β‐blockade in patients with CHF.