bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the ferrochelatase gene expression during differentiation of mouse erythroleukemia cells
Authors: Furukawa Takako , Tokunaga Rikio , Taketani Shigeru
Published: July 2007

Ferrochelatase [EC 4.99.1.1], the last step enzyme of heme biosynthesis, is transcribed from a single promoter. The enzyme is ubiquitously expressed in all cells, while the transcription is induced during erythroid differentiation. In a transient transfection assay, we identified two cis‐acting elements involved in regulating the human ferrochelatase gene in mouse erythroleukemia (MEL) cells; one is the distal region (‐108 to ‐95) for the basic expression in erythroid and non‐erythroid cells, and the other is the proximal region (‐80 to ‐72) corresponding to the induced expression during differentiation of dimethylsulfoxide‐treated MEL cells. DNase I footprinting and gel mobility shift analyses revealed the presence of nuclear protein binding to the distal and the proximal regions, in both induced and uninduced MEL cells. These mean that two promoter regions play an important role in regulating the induced expression of ferrochelatase.
Modulation of sperm acrosomal exocytosis by guanyl nucleotides and G‐protein‐modifier agents
Authors: Brandelli Adriano
Published: July 2007

Mammalian sperm must undergo an exocytotic event during fertilization, the acrosome reaction (AR). This process is specifically induced by egg‐surface glycoproteins and it involves guanine nucleotide binding proteins (G‐proteins). Neoglycoproteins (NGP) with mannose or N‐acetylglucosamine residues has been demonstrated to induce the AR in human sperm. Activators of G‐proteins, like GTPγS, GppNHp, mastoparan and A1F4‐ were capable of inducing the AR, while other nucleotides or analogues did not. When sperm were preincubated with these agents and then with NGPs, only the G‐protein inhibitor GDPIβS decreased the AR rate. The preincubation of sperm with Pertussis toxin resulted in the inhibition of NGP‐induced AR, while no effect was observed with cholera toxin. Results indicate that direct activation of G‐proteins is sufficient to elicit the AR, and the induction of the AR in human sperm is mediated by N‐acetylglucosaminyl and mannosyl binding sites involving PTx‐sensitive G‐proteins similar to the induction by zona pellucida glycoproteins.
Interaction of interferon‐γ and epidermal growth factor in the regulation of nitric oxide production and cellular proliferation in a cultured murine mammary cell line, COMMA‐D
Authors: Low Boon Chuan , Prosser Colin G. , Lacasse Pierre , Grigor Murray R.
Published: July 2007

The regulation of nitric oxide synthase (NOS) activity and gene expression by cytokines and growth factors has been studied using the murine mammary epithelial cell line, COMMA‐D. NOS activity was stimulated by exposure to interferon‐γ (IFN‐γ) and could be further stimulated by tumour necrosis factor‐α (TNF‐α) and epidermal growth factor (EGF) although neither was affective alone. The maximal activity observed in the presence of IFN‐α and EGF was not affected by the order in which cells were exposed. Messenger RNA levels for the inducible NOS isoform were increased by IFN‐γ and TNF‐α in a manner consistent with the elevation of NOS activity. EGF also stimulated thymidine incorporation into DNA which was attenuated by coexposure with IFN‐γ in a manner that appeared to be largely NO‐independent.
TsTX‐VII, a new toxin from Tityus serrulatus scorpion venom able to induce the release of neurotransmitters from rat brain synaptosomes not blocked by tetrodotoxin
Authors: Sampaio Suely V. , Coutinho‐Netto J. , Arantes Eliane C. , Toyama Marcos H. , Novello José C. , Giglio José R.
Published: July 2007

A procedure for the isolation of the toxin Tityustoxin VII (TsTX‐VII) from Tityus serrulatus scorpion venom and its biochemical characterization is reported. This protein has a Mr=6,700‐6,800, eight half‐cystine residues accounting for four disulfide bridges and no His. Its N‐terminal sequence GHZGYGS … characterizes it as a new toxin, able to release glutamic acid and gamma aminobutyric acid from rat brain synaptosomes “in vitro”. This release was also induced by the whole venom. Tetrodotoxin however blocked the effect of the whole venom but not that of TsTX‐VII, thus suggesting that the releasing mechanism by TsTX‐VII does not involve Na+ but perhaps K+ or Ca++ channels.
Enhanced in vivo catalytic activity of PEG‐modified cellulase complex from Trichoderma reesei
Authors: Mitra Gargi , Bachhawat Bimal K.
Published: July 2007

The cellulase complex from Trichoderma reesei was polyethylene glycol (PEG)‐modified with considerable retention of endo‐β‐1,4‐glucanase activity, as evaluated by the carboxymethylcellutase (CMCase) assay. While resistance towards heat denaturation was the same for either form, susceptibility towards proteolysis was slightly greater for the PEG‐conjugate, in contrast to most reports using other enzymes. The circulatory life of endoglucanase activity associated with the complex was enhanced upon PEG‐modification. This was confirmed by demonstrating degradation of chromogen‐tabelled substrate injected (i.v.) after 24hr of administration of the PEG‐ylated enzymes; in contrast the substrate remained undegraded in mice pretreated with the native complex. The PEG‐modified complex could be a good candidate for the treatment of pneumoconioses of textile workers exposed to cotton dust.
Isolation of the transferrin receptor from human placenta
Authors: Kanevsky Yu V. , Pozdnyakova L. P. , Katukov Yu V. , Severin S. E.
Published: July 2007

The transferrin receptor (TFR) has been detected in tissues characterised by a high degree of proliferation. We have developed a procedure for isolating TFR from human placental tissues by affinity chromatography on transferrin‐Sepharose. Using gel filtration and electrophoresis in 7% PAAG, it has been shown that the molecular mass of the protein is 180 kDa. The protein has a subunit structure and is made up of two identical subunits, 90 kDa each. The constant for the protein binding to transferrin is equal to 5×10‐9 M. The yield of the protein isolated by the novel procedure exceeds 5‐fold that obtained by previously described methods.
Inhibition of various isoforms of rat liver glutathione S‐transferases by tannic acid and butein
Authors: Zhang Kai , Mack Peter , Wong Kim Ping
Published: July 2007

Glutathione S‐transferases (EC.2.5.1.18, GSTs) were purified from rat liver by S‐hexylglutathione affinity chromatography and six isoforms, namely C‐1, C‐2, C‐3, C‐4, A‐2 and A‐1, were isolated by CM‐cellulose and DEAE‐cellulose ion‐exchange columns. Tannic acid and butein showed varying degrees of inhibition on the six individual GST isoforms. When 1‐chloro‐2,4‐dinitrobenzene (CDNB) was used as a substrate, butein exerted significantly more potent inhibition on the cationic isoforms C‐2, C‐3 and C‐4 with IC50 values of 6.8, 8.5 and 8.0 μM respectively. All the isoforms showed lower activity towards p‐nitrobenzyt chloride when compared to CDNB and inhibition of the p‐nitrobenzyl chloride‐activity by tannic acid and butein was also weaker. The inhibitory effects of tannic acid and butein on each isoform decreased generally with increasing pH in the range of 6.0 to 8.0. The optimum pHs for inhibitions by tannic acid and butein on the six individual isoforms lie in the pH range of 6.0 to 6.5.
Quantification of epididymal retinoic acid‐binding protein (ERABP) mRNA in different anatomical regions of the adult rat epididymis
Authors: Hall Joseph C. , Tubbs Christopher E.
Published: July 2007

Epididymal retinoic acid‐binding protein (ERABP), an 18.5 Kd androgen‐dependent protein, is synthesized and secreted by the rat epididymis. The concentration of ERABP mRNA in different anatomical regions of the rat epididymis was measured using quantitative reverse transcription polymerase chain reaction (RT‐PCR) and Northern blot analysis. While quantitative differences in the level of ERABP were observed in the caput region of the epididymis, no detectable levels of ERABP mRNA was observed in the corpus and cauda regions of the epididymis. In the caput region of the epididymis, 0.22 ng and 0.036 ng mRNA/gg of total RNA were measured in the proximal and distal segments of the caput region, respectively. These results indicate that ERABP is differentially expressed in the rat epididymis and suggest that the protein may be regulated at the level of transcription.
Stability of assembled epitopes on human chorionic gonadotropin to covalent modifications
Authors: Venkatesh N. , Murthy G. S.
Published: July 2007

A measure of stability of a given epitope is an important parameter in the exploration of the utility of a desired MAb. It defines the conditions necessary for using MAbs as an investigative tool in several research methodologies and therapeutic protocols. Despite these obvious interests the lack of simple and rapid assay systems for quantitating MAb‐Ag interactions has largely hampered these studies. A single step MAb‐Solid Phase Radioimmunoassay (SS‐SPRIA), is described which eliminates errors that may arise with multistep sandwich assays. SS‐SPRIA has been used to demonstrate the differential stability of the assembled epitopes on gonadotropins. Differential stability towards specific reagents can be exploited to identify aminoacid residues at the epitopic site. Inactivation of an epitopic region is indicative of the presence of the group modified, provided conformational relaxations are not induced due to modifications at distant sites. Here we provide evidence to validate these conclusions.
Isolation of intact transition protein 2 with three zinc finger motifs from boar late spermatid nuclei
Authors: Akama Kuniko , Sato Hiroki , Oguma Koichi , Nakano Minoru
Published: July 2007

Boar intact transition protein 2, TP2, was isolated from the late spermatid nuclei by chromatography on Fractogel EMD SO3‐ 650 (M), and HPLCs on Nucleosil 300 7C18, Diol‐120 and Chemcosorb 3C18H. CD spectroscopy study showed that TP2 underwent a small but significant zinc dependent secondary structural change. TP2, having three potential zinc finger motifs, was shown to be contain 3 atoms of zinc bound per molecule of the protein by atomic absorption spectroscopy. These results together with the amino acid sequence of TP2 suggest that TP2 is a zinc metalloprotein with three zinc finger structures.
Fusion of phospholipid vesicles induced by the ribosome inactivating protein saporin
Authors: Liu Guohong , Hao Qiang , Zhang Yah , Gao Gui , Yan Ganglin , Yao Qizhi , Li Qingshan
Published: July 2007

The single chain ribosome‐inactivating protein Saporin‐S6 (SO‐6) induces the fusion of acid phospholipid vesicles. The extent of fusion was measured by resonance energy transfer assay between the N‐(7‐nitro‐2‐1,3‐benzoxadiazol‐4‐yl)‐dimyristoylphosphatidyl lithanolamine (NBD‐PE)(donor) and N‐(lissamine rhodamine B sulphonyl)‐diacylphoshaidylethanolamine (Rh‐PE) (acceptor) incorporated in the vesicle. The saturated lipid/protein molar ratio is approx. 100:1. The time course of fusion of vesicles induced by the protein showed that the process was completed within 10 minutes, and the size of the particles in the medium was enlarged which conforms the occurrence of the fusion occuring. The fusion is temperature dependent and the liquid‐crystalline state lipid is more apt to fuse than the gel phase lipid. The effect of SO‐6 is also dependent on ionic strength and pH, high salt concentration and basic pH may abolish fusion, which suggests that both electrostatic and hydrophobic components may be involved in the process.
The transcriptional repression of the human Cu/Zn superoxide dismutase(sod1) gene by the aticancer drugg, mitocycin C(MMC)
Authors: Cho Ginam , Kang Sangsun , Seo Seong Jin , Kim Younhee , Jung Guhung
Published: July 2007

The Cu/Zn superoxide dismutase(sod1) is one of the key enzymes that protects cells against oxidative stress. In order to investigate the effects of mitomycin C(MMC) on the induction of apoptotic cell death and on the sod1 transcription level, the CATs activity of HepG2 cells transfected with sod1 promoter‐CAT(chloramphenicol acetyl transferase) fusion reporter was measured after MMC treatment. The CAT assay showed that exposure of HepG2 cells to MMC decreased the transcription level of the sod1 gene. The accumulation of p53 tumor suppressor protein by MMC treatment of HepG2 cells was noted. In order to investigate the p53‐negative response element in its promoter region, a p53 cotransfection experiment with serially deleted sod1 promoter/CAT reporter constructs was performed. The results show a significant reduction of CAT activity in all deletion reporter constructs. The results show that MMC treatment inhibited sod1 gene transcription through p53‐mediated transcriptional repression.
Autolytic site mutant R105C of rat trypsin
Authors: Li Xin‐Fang , Tang Jian‐Guo , Zhang Long‐Xiang
Published: July 2007

The autolytic site Argl05 of rat trypsin was replaced with Cys by DNA site‐directed mutagenesis method. Comparison of expression and purification of R105C trypsin along with the wild type and some other Arg105 mutants indicates that R105C trypsin could be expressed as well but with a lower expression level. It is unexpected that R105C trypsin has no detectable activity toward trypsin substrate TAME, quite different from the wild type and other Argl05 mutants. Native gel electrophoresis analysis indicates that R105C trypsin has a similar mobility rate to that of wild type trypsin. FPLC also gives similar retaining time. The loss of activity of R105C trypsin may result from the conformational change around active site, but not the dimer formation.
Oxidized low‐density‐lipoprotein triggers programmed death of endothelial cells
Authors: Seelos Christian , Gmeiner Bernhard , Kapiotis Stylianos , Cerni Christa
Published: July 2007

Incubation of bovine aortic as well as human umbilical vein endothelial cells with either oxidized or native low‐density‐lipoprotein in the presence of trace amounts of copper induced morphological changes of the cells and chromatin fragmentation characteristic for programmed cell death. Shrinkage of cells was evident after 6 to 8 hours of incubation and clearly preceded release of lactate dehydrogenase as a marker of cell permeability. Condensation of nuclear chromatin and internucleosomal cleavage was demonstrated by Hoechst staining and gel electrophoresis, respectively. Thus, by inducing active death of endothelial cells oxidized low‐density‐lipoprotein might negatively influence tissue homeostasis of the endothelium and thereby promote the development of atherosclerotic plaques.
Occurrence of nonenzymatic N‐acetylation of sphinganine with acetyl coenzyme a producing C2‐H2‐ceramide and its inconvertibility to apoptotic C2‐ceramide
Authors: Kashiwagi Motoi , Mikami Takeshi , Chiba Masahiko , Chiba Susumu , Matsumoto Hiroyuki , Akino Toyoaki , Gasaa Shinsei
Published: July 2007

Sphinganine, a biosynthetic precursor of ceramide, was non‐enzymatically acetylated with acetyl coenzyme A at the C‐2‐amino residue to produce C2‐H2‐ceramide (N‐acetyl sphinganine) in an organic solvent and in an aqueous solution with a high yield, where as sphingenine was only acetylated slightly. The structure of the N‐acetyl sphinganine was identified with mass spectrum, and with chromatography using an authentic N‐acetylated substance. Furthermore, the C2‐H2‐ceramide was examined for enzymatic desaturation to determine whether C2‐ceramide, a cell‐permeable ceramide responsible for apoptosis of cells, was produced, revealing an inferior substrate for H2‐ceramide desaturase of horse brain microsomes.
Identification, purification, and partial characterization of a factor from rabbit serum that inhibits prolactin secretion by pituitary cells cultured in vitro
Authors: Giacomelli Sabrina , Cheng C. Yan , Leone Maria Grazia , Palmery Maura , Silvestrini Bruno
Published: July 2007

A biological factor that inhibited prolactin secretion by pituitary cells cultured in vitro was identified, purified, and partially characterized from normal rabbit serum. This biological factor was also found to potentiate dopamine‐mediated aortic contraction using rabbit aortic strips in vitro. Following SDS‐PAGE, this factor displayed an apparent Mr of 17 kDa, which is different from the Mr of most known endogenous factors having an inhibiting activity on pituitary prolactin secretion, suggesting that this may be a yet‐to‐be identified novel molecule.
Characterization of the orf31‐petG gene cluster from the plastid genome of Populus deltoides
Authors: Naithani Sushma , Trivedi Prabodh K. , Sane Prafullachandra V.
Published: July 2007

The orf31‐petG gene cluster is located approximately 1.2 kb away from the psbEFLJ operon in the chloroplast genome of Populus deltoides. The orf31 (ycf7 encodes an unidentified polypeptide while the petG gene encodes subunit V of an important component, cytochrome b6/f complex, involved in photosynthetic electron transport. We have determined the nucleotide sequence of the orf31‐petG gene cluster from the plastid geneme of a tree, Populus deltoides. Our sequence analysis suggests that these genes possess high homology with the published sequences of these genes from other plants. Northern analysis suggests development dependent transcription of the orf31‐petG cluster in leaves.
Comparison of SSCP analysis and CFLP analysis for mutation detection in the human iduronate 2‐sulfatase gene
Authors: Maddox Lewis O. , Li Peining , Bennett Amy , Descartes Maria , Thompson Jerry N.
Published: July 2007

Scanning methodologies are used for the identification of DNA fragments that differ from the normal nucleotide sequence. Fragments that produce abnormal band patterns are sequenced for characterization of the exact mutation. Factors considered in choosing a scanning methodology include reproducibility, sensitivity, and time. In the present study, we compared single‐stranded conformational polymorphism (SSCP) and Cleavase fragment length polymorphism (CFLP) methodologies for mutation scanning of exon VIII in the iduronate 2‐sulfatase (IDS) gene. Mutations of the IDS gene result in an X‐linked lysosomal storage disease, Hunter syndrome. These six known mutations analyzed by the two methods included a one base pair deletion, a one base pair insertion, and four point mutations. SSCP analysis detected all of the mutations and CFLP analysis detected three of the six mutations. We concluded that SSCP analysis was preferable to CFLP analysis for scanning exon VIII in the IDS gene for mutations.
The differential in vitro stimulation of 3′,5′‐cyclic nucleotide phosphodiesterase by calcium binding proteins
Authors: La Bella Vincenzo , Ho Bao‐Kuang , Appel Stanley H.
Published: July 2007

A eterogeneous class of proteins exhibit within their sequence a particular structure, named EF‐hand, able to bind calcium with high affinity. These calcium binding proteins have been described in most cells and tissues and are suggested to work as calcium buffers, thereby participating in the regulation of calcium‐dependent cellular activity. Recent circumstantial evidences suggest that calcium binding proteins may serve other functions as well, possibly as enzyme modulators. Since 3′,5′‐cyclic nucleotide phosphodiesterase is a well‐known calmodulin‐modulated enzyme, in this work we studied the effect in vitro of different purified calcium binding proteins on the activity of this enzyme. Among the proteins tested, calmodulin and recombinant rat brain parvalbumin could stimulate the 3′,5′‐cyclic nucleotide phosphodiesterase activity in vitro, whereas rabbit muscle parvalbumin, rat renal and brain calbindin D28K, and bovine brain S‐100B were ineffective. Immunoprecipitation with the specific antiserum completely abolished either calmodulin or recombinant brain parvalbumin activation of 3′,5′‐cyclic nucleotide phosphodiesterase. Moreover, while the presence of calcium in the incubation mixture was critical in the calmodulin‐mediated stimulation of the enzyme, it did not modify the effect of the recombinant brain parvalbumin. We suggest that, in addition to calmodulin, parvalbumin may be a regulator of 3′,5′‐cyclic nucleotide phosphodiesterase, and possibly of other yet to be identified enzymes in certain tissues.
Biochemical properties of hemagglutinins in the mollusk Pomacea flagellata
Authors: Arreguín‐Espinosa Roberto , Arreguín‐Lozano Barbarín
Published: July 2007

Extracts of the Mollusk Pomacea flagellata were found to contain two agglutinins (PFA‐I and PFA‐II) which reacted with erythrocytes from human and several animal species. The hemagglutinating activity was stable between pH 4.0 and pH 10.0, and was independent of divalent cations. The active components were purified and found to be glycoproteins having Mr of 30,000. The isoelectric points were estimated to be 4.7 and 4.9 for PFA‐I and PFA‐II respectively. Hapten inhibition assays indicated that the agglutinin and its isoforms are specific for β‐galactosides as well as for galactose containing glycoproteins.
Conformational changes of creatine kinase in trifluoroethanol solutions
Authors: Yang Hai‐Peng , Zhou Hai‐Meng
Published: July 2007

The conformational changes of creatine kinase (ATP:creatine N‐phosphotrnsferase, EC 2.7.3.2) in trifluoroethanol solutions have been followed by fuorescence emission and circular dichroism spectra. At low trifluoroethanol concentrations, less than 15%, the enzyme was completely inactivated with no observed marked conformational changes. The fluorescence emission maximum of the native enzyme was at 337 nm. With increasing trifluoroethanol concentration, the fluorescence emission maximum red‐shifted in magnitude to a maximum value (355nm) at 40% trifluoroethanol, indicating that the tryptophan residues were completely exposed. The results obtained from CD spectra show that the presence of trifluoroethanol can induce the formation of secondary structure in the native enzyme and in urea‐denatured enzyme.
Enhanced expression of initiator tRNAMet in human gastric and colorectal carcinoma
Authors: Kanduc Darja , Lucchese Alberta , Corcia Maria Grazia di , Natale Costanzo
Published: July 2007

Transfer RNA isoaccepting species are differentially expressed at different times during development, differentiation, growth, aging, and carcinogenesis processes. It has been suggested that alterations in tRNA patterns might be mechanistically important in modulating gene expression during the various physiological/pathological cellular stages. As part of a study to investigate the possible mechanisms by which alterations of translational machinery can start and/or sustain carcinogenic cell proliferation, in this communication we report analysis of tRNA distribution in two gastro‐intestinal human tumors. The qualitative and quantitative data obtained for cellular tRNA distribution put into evidence a shift in the tRNA population with increased level of initiator tRNAMet in the malignant tissues. This observation confirms previous data obtained on experimental carcinogenesis models and suggests the possibility of specific involvement of tRNA changes in protein synthesis initiation during tumorigenesis.
The role of acetic acid in the prevention of salt‐induced aggregation of snake venom cardiotoxins
Authors: Sivaraman T. , Kumar T. K. S. , Huang C. C. , Yu C.
Published: July 2007

Snake venom cardiotoxins (CTXs) exhibit a strong tendency to aggregate upon desalting and hence it is extremely difficult to prepare salt‐free cardiotoxin(s). In the present study, we describe a new method for preparation of salt‐free CTX based on dialysis against acetic acid. Based on experimental observation and the three dimensional solution structure of cardiotoxin analogue III from the Taiwan cobra (Naja naja atra), a molecular mechanism for the prevention of aggregation of cardiotoxins by acetic acid is discussed. In our opinion, the results obtained in the present study would pave way for elucidating the structural basis for the broad spectrum of biological activities exhibited by snake venom cardiotoxins.
Compromised optimal hybridization temperatures may explain PM plus DQA1 anomalies
Authors: Riley Donald E.
Published: July 2007

The multiplex DNA typing test kit, PM plus DQA1TM relies on 36 different olignucleotides that all must hybridize correctly for accurate DNA typing. The G+C contents of the probe oligonucleotides range from 47% to 83%. Since G+C content directly affects optimal hybridization temperatures, a wide range of optimal hybridization temperatures is expected. This was verified by two empirically tested formulas. The recommended hybridization temperature is 55°C. The control probe has an estimated optimal temperature of 75°C indicating low stringency of the control. The B allele probe of the GC locus has an estimated optimal hybridization temperature of 70°C. These discrepancies are unexplained by GC clusters or sequence palindromes. The departures from temperature optima may be responsible for observed PM plus DQA1 artifacts and for previously observed, low tolerance of this system to temperature variation. Since optimal temperature and cation concentration are related variables, careful attention to these is recommended as are positive controls demonstrating amplification of all alleles monitored by PM plus DQA1.
A surface plasmon resonance assay for the binding of Amaranthus hypochondriacus var. Mexico lectin to glycoprotein
Authors: Ozeki Munetaka , Furuichi Yukio , Umekawa Hayato , Takahashi Takao
Published: July 2007

We have used the analytical system based on surface plasmon resonance to monitor the interaction between Amaranthus hypochondriacus var. Mexico lectin and four different fetuins; fetuin, asialofetuin, agalactofetuin, and agalactosaminofetuin. Agalactofetuin and agalactosaminofetuin were prepared by enzymic digestion of asialofetuin using jack bean β‐galactosidase or endo‐α‐N‐acetylgalactosaminidase from Diplococcus pneumoniae. Ligands were immobilized onto a sensor surface via amide linkages. The lectin interacted most strongly with asialofetuin, but not with agalactosaminofetuin. The binding of the lectin to asialofetuin was inhibited by N‐acetylgalactosamine or Galβ1→3GalNAc in a dose‐dependent manner.
Increment of efficiency in the identification of noble genes by colony hybridization assay
Authors: Kim J. W. , Lee I. A. , Lee Y. H. , Song J. C. , Choe Y. K. , Hahn Y. S. , Chung J. H. , Chung T. W. , Choe I. S.
Published: July 2007

For the rapid identification of noble genes in a specific tissue by computer analysis from the cDNA sequences determined by single‐pass cDNA sequencing, clone redundancy was one of the major obstacles. To facilitate the efficiency in identification of noble genes, it was necessary to reduce the number of clones to be sequenced by eliminating the redundant clones for a rapid analysis. In order to increase the probability of isolating noble sequences from the cDNA clones of human fetal liver tissue origin, colony hybridization assay was adopted and redundant clones were efficiently removed. Four cDNA clones highly redundant in the human fetal liver cDNA libraries including α‐globin, γ‐globin, serum albumin and H19 RNA sequences were selected as the probes. Two hundreds and sixty two cDNA clones were randomly selected and tested with the probes for hybridization properties. The identity of each cDNA clone giving positive or negative signals in the hybridization assay was determined by DNA homology search with the nucleic acid databases. Among the 76 clones giving positive signals, 57 clones (75%) were found to be identical to the probe sequences and could be eliminated by colony hybridization assay before neucleotide sequencing.
Mapping of quantitative trait loci for body weight on chromosomes 1 and 4 in the rat
Authors: Kovács Peter , Klöting Ingrid
Published: July 2007

Quantitative trait loci (QTLs) affecting body weight were investigated in the backcross population derived from diabetic BB/OK and spontaneously hypertensive rat (SHR). The F1 hybrids were backcrossed onto BB/OK rats, and QTL analysis was performed with the resulting backcross population on chromosomes 1, 3, 4, 10, 13 and 18. According to the stringent threshold for a lod score of 3.0, markers on chromosomes 1 and 4 were found to be linked with body weight. The QTL with a peak lod score (3.3) on chromosome 1 for a male population was located within the region flanked by loci Igf2 and D1Mgh12. On chromosome 4, linkage between the body weight and the region around the Npy locus was observed (lod score 3.1). The existence of the QTL on chromosome 4 affecting body weight was confirmed by congenic BB.LL rats, carrying chromosomal region of SHR (D4Mit6‐Npy‐Spr) on the genetic background of the BB/OK rat.
Isolation and characterization of CspBI, a novel NotI isoschizomer from Corynebacterium species B recognizing 5′‐GC↓GGCCGC‐3′
Authors: Sokolov Nikolai N. , Eldarov Michael A. , Rina Mary , Korolev Sergey V. , Markaki Maria , Kalugin Alexei A. , Omelyanuk Natalia M. , Skryabin Konstantin G. , Bouriotis Vassilis
Published: July 2007

Sixty‐seven bacterial strains were surveyed for the presence of type II restriction endonucleases, especially concerning super‐rare‐cutting enzymes. Fourteen strains were found to contain specific enzymes. One of them CspBI from Corynebacterium species B was purified and characterized as an isoschizomer of NotI, which recognizes the palindromic octanucleotide sequence 5′‐GC↓GGCCGC‐3′ and cleaves at the position shown by the arrow. A comparison between the cleavage patterns on different DNAs, obtained with partially purified endonucleases from other detected producents including some strains of Corynebacterium, Cellulomonas and Rhizobium has shown that these enzymes do not belong to super‐rare‐cutting restriction endonucleases.
Cross‐linking induces homodimer formation and inhibits enzymatic activity of chicken stomach ecto‐aryrase
Authors: Carl Stephanie Ann Lewis , Smith Thomas M. , Kirley Terence L.
Published: July 2007

We have investigated the effect of cross‐linking on the enzymatic activity and oligomer formation of the chicken stomach ecto‐apyrase. Cross‐linking with the hydrophobic, lysine‐specific dithiobis(succinimidylpropionate) (DSP) caused equal inhibition of ATPase and ADPase activity in both the membrane‐bound and detergent‐solubilized ecto‐apyrase. The inhibitory effect of cross‐linking was reversed upon the addition of the reductant dithiothreitol. Western blots of aliquots of the cross‐linked samples show decreased amounts of the monomeric 80 kDa ecto‐apyrase and the appearance of a 160 kDa dimer under conditions inducing enzyme inhibition. Therefore, the chicken stomach ecto‐apyrase, like the chicken gizzard ecto‐ATPase, is likely a homodimer in vivo. Unlike the related gizzard ecto‐ATPase, however, the native stomach ecto‐apyrase is not stimulated, but rather inhibited by cross‐linking, presumably due to different quaternary structural stability of the two enzymes.
Carbohydrates regulate the dimerization of angiotensin‐converting enzyme
Authors: Kost Olga A. , Orth Tatyana A. , Nikolskaya Irina I. , Nametkin Sergei N. , Levashov Andrey V.
Published: July 2007

Summary. Regulation of the catalytic activity and supramolecular structure of angiotensin‐converting enzyme was studied in reverse micelles of Aerosol OT in octane as biomembrane model. The kinetic experiments and the sedimentation analysis demonstrated that the enzyme can function both in monomeric and dimeric form. The degree of dimerization was strongly dependent on the concentration and structure of mono‐ and disaccharides added to the media, indicating the specific role of carbohydrates in forming the supramolecular structure of angiotensin‐converting enzyme. The existence of carbohydrate‐binding center on the enzyme molecule is proposed.
Saccharide‐assisted delivery of cytotoxic liposomes to human malignant cells
Authors: Vodovozova Elena L. , Gayenko Galina P. , Razinkov Vladimir I. , Korchagina Elena Yu. , Bovin Nikolai V. , Molotkovsky Julian G.
Published: July 2007

The overexpression of lectins by malignant cells was applied for in vitro targeting of liposomes equipped with a saccharide vector and loaded in the lipid phase with a lipid derivative of anticancer agent sarcotysine. The lectin specificity of human leukemia HL‐60 and human lung adenocarcinoma ACL cells was revealed by tests with fluorescein‐labeled sugar probes. With the help of fluorescent lipid dye it was shown that active saccharide ligands increased the level of the vectored liposome binding to malignant cells by 50‐80% as compared to liposomes without vector or with inactive one. The degree of liposome/cell membrane fusion was monitored fluorometrically and was shown to be complete and independent of the vectors. The targeted drug‐loaded liposomes had the cytotoxic activity 2‐4 times higher as compared to the vector‐free ones.
Investigation of the Yeast Mitochondriai Unselective Channel in intact and permeabilized spheroplasts
Authors: Manon Stéphen , Guérin Martine
Published: July 2007

The existence of an activity corresponding to the nucleotide‐induced Yeast Mitochondria Unselective Channel (YMUC2) of isolated mitochondria was investigated in permeabilized and intact spheroplasts of the baker's yeast Yeast Foam. In nystatin‐permeabilized spheroplasts, ATP and GDP‐β‐S induced a decavanadate‐sensitive stimulation of the respiration only under conditions equivalent to those previously reported for isolated mitochondria (low phosphate concentration, presence of a salt). On intact spheroplasts, parallel measurements of respiration rate, [ATP]/[ADP] ratio and mitochondrial transmembrane potential allowed to show that the addition of the glucose analog 2‐deoxyglucose decreased the permeability of the inner mitochondrial membrane owing to cellular ATP depletion. This strongly supports the hypothesis that Yeast Mitochondria Unspecific Channel is active in situ and inhibited by cellular [ATP] depletion.
Absorption and fluorescence spectra of polyene antibiotics in the presence of human serum albumin
Authors: Romanini Diana , Farruggia Beatriz , Picó Guillermo
Published: July 2007

The alteration in the fluorescence spectra observed for the polyene antibiotics: nystatin and amphotericin B in the presence of human serum albumin is due to a decrease in the polar character of the antibiotic environment when these are bound to the protein. Amphotericin B showed two types of binding sites, the first having very high affinity (5.8 107 M‐1) and a secondary binding site with an affinity one order lower than the primary sites. This secondary binding site was very sensitive to temperature change. Nystatin yielded only one type of binding sites with an affinity of 1.1 106 M‐1. An electrostatic component was found in the binding of both ligands, as well as an important disorder at the protein binding sites. However the secondary binding site for AMP showed negative entropie change value, which suggests different mechanim of binding respect to the primary one. Conformational change induced by the temperature in the albumin molecule was detected by nystatin binding. Fatty acids produced an interference in the binding of both antibiotics to albumin.
Genetic variants of chicken erythrocyte histone H5
Authors: Górnicka‐Michalska Ewa , Pałyga Jan , Lubon Henryk , Kowalski Andrzej , Cywa‐Benko Katarzyna
Published: July 2007

Two allelic electromorphs a and b of chicken eryhrocyte histone H5 have been detected in a sodium dodecyl sulfate polyacrylamide gel. In an acid‐urea gel, however, each of the allelic variants was found to be accompanied by a slower migrating form. A comparison of α‐chymotrypsin‐digested products of H5.a and H5.b revealed that they differed in N‐terminal domains. The H5 variants were distributed differently not only in various chicken races but also in distinct lines within a breed. Allele H5b was about 2.6‐4.6 as abundant as its counterpart H5a in most chicken populations examined. These proportions were distorted in two Leghorn lines: the ratio of H5b to H5a was only 1.6 in line H22 and increased up to 32 in line G99.
Esterification of D‐mannoheptulose confers to the heptose inhibitory action on D‐glucose metabolism in parotid cells
Authors: Malaisse Willy J. , Kadiata Marcel M. , Scruel Olivier , Sener Abdullah
Published: July 2007

D‐mannoheptutose, but not its hexaacetate ester, inhibits, in a competitive manner, D‐glucose phosphorylation by either purified beef heart hexokinase or crude parotid gland homogenates. Yet, D‐mannoheptulose hexaacetate, but neither the unesterified heptose nor acetate or its methyl ester, inhibits D‐[5‐3H]glucose utilization and D‐[U‐14C]glucose conversion to 14CO2 and 14C‐labelled acidic metabolites aud anaino acids in intact isolated parotid cells. It is proposed, therefore, that D‐mannoheptulose hexaacetate crosses efficiently the plasma membrane of parotid cells and, after intracellular hydrolysis, allows inhibition of D‐glucose phosphorylation by the unesterified heptose. The ester of D‐mannoheptulose could thus represent a useful tool to inhibit hexose phosphorylation and interfere with cell growth in cells otherwise resistant to the heptose.
Hepatic heterogeneity in the response to AMP studied in the bivascularly perfused rat liver
Authors: Minguetti‐Câmara Vania Cibele , Constantin Jorgete , Suzuki‐Kemmelmeier Fumie , Bracht Adelar
Published: July 2007

The zonation of the purinergic action of AMP in the hepatic parenchyma was investigated in the bivascularly perfused rat liver by means of anterograde and retrograde perfusion. Livers from fed rats were used and AMP (100 μM) was infused according to four different experimental protocols: (A) anterograde perfusion and AMP infusion via the portal vein; (B) anterograde perfusion and AMP infusion via the hepatic artery; (C) retrograde perfusion and AMP via the hepatic vein; (D) retrograde perfusion and AMP via the hepatic artery. The response of the liver cells was heterogeneous. Oxygen uptake inhibition by AMP predominates in cells situated shortly after the intrasinusoidal confluence of the portal vein and hepatic artery. Oxygen consumption in all other cells seems to be increased by AMP. Glycogenolysis stimulation by AMP (glucose release) was more pronounced in the periportal cells situated in the region of the intrasinusoidal confluence of the portal vein and the hepatic artery. It can be concluded that the heterogenic response of the liver to AMP is similar to the heterogenic response to ATP.
Effect of salts on the interaction of oxidized glutathione with dibasic amino acids
Authors: Cserháti Tibor
Published: July 2007

The interaction of oxidized glutathione (GSSG) with dibasic amino acids was studied with charge‐transfer chromatography carried out on unimpregnated cellulose layers in the absence and presence of LiCl, NaCl, KCl, MgCl2 and CaCl2. GSSG decreased in each case the apparent lipophilicity of dibasic amino acids, indicating some type of interaction. Calculations proved that the strength of GSSG ‐ dibasic amino acid interaction decreases with increasing concentration of salts suggesting a hydrophilic character of interaction. It can be assumed that electrostatic forces between the carboxyl group of GSSG and the amino groups of amino acids are involved in the interaction. GSSG binds stronger to arginine than to lysine and ornithine suggesting that arginine is probably the primary binding site in proteins for GSSG.
Fatty acid‐mediated uncoupling of potato tuber mitochondria
Authors: Saviani Elzira E. , Martins Ione S.
Published: July 2007

The present work examined whether the ATP/ADP carrier, other than the plant uncoupling mitochondrial protein, participates in free fatty acid‐mediated uncoupling of potato tuber mitochondria. The basal respiration rate of succinate‐energized mitochondria was stimulated by a low concentration of palmitate (20μM). This uncoupling was reversed by 10μM carboxyatractyloside and by the subsequent addition of 0.1% bovine serum albumin. The decrease in membrane potential caused by palmitate was suppressed by carboxyatractyloside (1μM) and, to a lesser degree, by bongkrekate (20μM). GTP could also reversed this decrease via a carboxyatractyloside‐independent mechanism. These results indicate that the ATP/ADP carrier, along with the plant uncoupling mitochondrial protein, participates in the protonophoric action of palmitate in potato tuber mitochondria.
The interaction of alcohol peroxyl radicals with human oxyhemoglobin
Authors: Szweda‐Lewandowska Z. , Krokosz A. , Zajaczkowska W. , Puchaŀa M.
Published: July 2007

The effects of interaction of ethanol, l‐butanol or t‐butanol peroxyl radicals with oxyhemoglobin (HbO2) were estimated using absorption spectroscopy in the visible range and tryptophan fluorescence. Peroxyl radicals were generated by gamma‐radiation. Alcohol peroxyl radicals showed the same effectiveness in oxidizing HbO2 iron as ‐OH radicals (GFe(III) = 1.8 for a dose 0.33 kGy). However, they degraded hemoglobin to hemichromes and cholehemichromes to a lesser degree than ‐OH radicals. In addition l‐ and t‐butanol peroxyl radicals were less effective than peroxyl ethanol radicals. Alcohol peroxyl radicals caused unfolding of protein to a much lesser degree than ‐OH radicals. Their contribution to the destruction of tryptophan residues was nearly matched by those obtained with the ‐OH radicals.
Synthesis of a unique polysialoganglioside by polysialyltranfferase from embryos of sea urchin Lytechnius pictus: Size determination of polysialic acid chain synthesized onto GD3 in vitro
Authors: Cho Jin Won
Published: July 2007

It has been reported that polysialyltransferase in sea urchin embryos polysialylates 38 kDa endogenous acceptor proteins and disialyllactose moieties of mammalian ganglioside GD3, and its maximum activity was observed at gastrula stage using an excess of GD3 as an exogenous acceptor (1). The objective of the present investigation is to determine how many sialic acid residues are transferred onto the disialyllactose moiety of GD3 by the polysialyltransferase when it is used as an exogenous acceptor. Ceramide glycanase which can cleave the linkage between ceramide and sugar moiety was treated to obtain hydrophilic polysialyllactose from polysialylated GD3. Surprisingly, size analysis of these chains using non‐SDS polyacrylamide gel electrophoresis showed that up to 60 sialic acid residues were transferred to the disialyllactose moiety of GD3 by polysiatyltransferase from the sea urchin embryos.
Human DNA‐topoisomerase I activity is affected by bis‐netropsin's binding to DNA minor groove
Authors: Sukhanova Alyona , Grokhovsky Sergei , Zhuze Alexei , Roper David , Bronstein Igor
Published: July 2007

Bis‐netropsins (bis‐Nts) are known to be efficient inhibitors of human DNA topoisomerase (topo) I with a higher antitumor activity than netropsin. New sequence‐specific derivatives of bis‐Nts were used for modulation of topo I‐mediated DNA cleavage with and without camptothecin (CPT). Relation between the bis‐Nts binding sites and topo I cleavage sites has been analyzed with the plasmid DNA constructs generated by insertion of synthetic oligonucleotides containing various topo I‐cleavage and bis‐Nt‐binding sites. These constructs offer an opportunity to study minor groove binders effects on the topo I reaction on DNA. Three major effects: (i) bis‐Nt ‐ mediated disappearance of some of the topo I cleavage sites; (ii) enhancement of some other sites, and, (iii) generation of new cleavage sites, have been fovnd and analyzed. These effects demonstrate that bis‐Nts drastically change the topo I‐mediated DNA cleavage.
Cloning and sequence analysis of novel cardiotoxins/CLBP from venom glands of Jaja naja atra
Authors: Chu Ren‐Chian , Yang C. C.
Published: July 2007

Seventeen cardiotoxins (CTXs)3 and four CLBPs3 have been identified by screening cDNA library and in conjunction with the PCR approach. Among the 17 CTXs, two have silent substitutions, five have previously been identified by traditional protein sequencing methods. In addition, ten not well known CTX genes and one new CLBP have been identified. These CTX genes show very high sequence homology with the major variations occurring at the tip of each individual loop. Although the CTX isoforms differ by only one or few residues and this subtle differences strongly influences the structure and function of the CTX isoforms. We have identified a novel CTX‐3c sequence which represents a blend of the CTX‐1 and CTX‐3 sequence. Based on the sequence comparison with other CTX isoforms from the same snake source, we predict interesting functional properties for CTX‐3c.
The interaction of phospholipase A2 with liposomes: An immunological approach to its study
Authors: Morales Rogelio , Zavala Eunice , Fernández Marta S.
Published: July 2007

Porcine pancreatic phospholipase A2 (PLA2) has been incubated for 2 hours at 34°C with rabbit anti‐pig pancreatic PLA2 polyclonal antiserum in the absence or presence of phosphatidylcholine liposomes in different physical states. Subsequent assay of hydrolysis ‐triggered through addition of 5 mM Ca2+‐ at 34°C, shows that preincubation with antiserum in the presence of 1,2‐dimyristoyl‐3‐sn‐phosphatidylcholine liquid‐crystalline vesicles, renders the PLA2 activity undetectable, similarly to what is found if preincubation is carried out in the absence of liposomes. In contrast, 1,2‐dipalmitoyl‐3‐sn‐phosphatidylcholine liposomes, which at 34°C are in the gel‐phase, protect the enzyme from the antiserum effect. The results are consistent with a stronger binding of PLA2 to gel phase, as compared to liquid crystalline vesicles and suggest that through the physical interaction with liposomes in the gel state, the enzyme is shielded from reaction with the antibodies. Taking into account the characteristic hydrolysis profiles of vesicles in different physical states, it can be concluded that the above interpretation agrees with the proposal that PLA2‐membrane association promotes the interfacial activation of the enzyme.
Molecular cloning and regulation of a novel guinea pig cytochrome P450 (CYP3A20) which differs from guinea pig CYP3A14 in only two amino acid residues
Authors: Mori Toshiyuki , Itoh Susumu , Kamataki Tetsuya
Published: July 2007

A full length cDNA clone of cytochrome P450, encoding 503 amino acid residues, was isolated from a male guinea pig liver cDNA library. The sequence was highly homologous to other members in the CYP3A subfamily and was designated CYP3A20. CYP3A20 and CYP3A14, another guinea pig CYP3A, shared 99.4% nucleotide and 99.6% deduced amino acid sequence homology. There were only two amino acid differences between CYP3A20 and CYP3A14. No significant induction of CYP3A20 mRNA in the livers from male guinea pigs treated with dexamethasone was observed by S1 mapping analysis although CYP3A14 mRNA was induced. The expression of CYP3A20 mRNA in the livers did not change between 5 and 10 weeks after birth while that of CYP3A14 mRNA in livers significantly increased at 10 weeks. This is the first report that the two highly resembling forms of cytochrome P450 display differently regulated expression from each other.
Characterisation of L‐[3H]glutamate binding to fresh and frozen crude plasma membranes isolated from cerebral cortex of adult rats
Authors: Emanuelli T. , Antunes V. F. , Souza D. O. G.
Published: July 2007

Specific [3H]glutamate binding to fresh crude plasma membranes (CPMs) was compared with binding to frozen CPMs and the optimal conditions for the binding to frozen CPMs isolated from cerebral cortex of adult rats were determined. Freezing reduced [3H]glutamate binding (3.5‐fold), and pre‐incubation of previously frozen membranes followed by three washes increased binding (4.5‐fold) when compared to fresh samples. CPMs washed once, pre‐incubated at 37°C and washed 3 times was adopted as the most adequate condition for the binding assay of frozen membranes. In a Cl‐‐containing medium, [3H]glutamate binding (Bmax=97.9 pmol/mg, Kd=349.68 nM) to this frozen CPM preparation was significantly displaced by excess quisqualic acid (QA) (65%), L‐2‐amino‐4‐phosphonobutyric acid (L‐AP4) (35%), trans‐1‐aminocyclopentane‐1,3‐dicarboxylate (1S,3R‐ACPD) (25%) and alfa‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) (25%). In a Cl‐‐free medium, binding (Bmax=44.14 pmol/mg, 311 nM) was significantly displaced by QA (45%), L‐AP4 (25%), ACPD (25%), AMPA (25%), kainic acid (20%) and N‐methyl‐D‐aspartate (15%).