bluuebunny/crossref_metadata_2025 huggingface dataset:
An increase in the carbohydrate moiety of α2‐macroglobulin is associated with systemic lupus erythematosus (SLE)
Authors: Panzironi Claudio , Mo Meng‐yun , Mruk Dolores , Cheng C. Yan , Silvestrini Bruno , Lahita Robert
Published: July 2007

Using lectin blots in conjunction with peptide mapping, α2‐macroglobulin micropurified from systemic lupus erythematosus (SLE) patients was shown to become abnormally glycosylated suggesting the occurrence of complex glycosylation in this pathological condition. To confirm there is indeed a quantitative increase in specific monosaccharides in this protein; α2‐macroglobulin was micropurified from a battery of 37 serum samples which included 6 normal donors (3 male and 3 female), 23 SLE patients, 6 rheumatoid arthritis patients, 1 mixed connective tissue disease patient, and 1 Sjogren's syndrome patient; for carbohydrate analysis. It was noted that the concentration of total monosaccharides in α2‐macroglobulin micropurified from serum samples of SLE patients is significantly higher than normal donors with a mean±SD of 188±410 μg/mg protein (SLE, n=23) versus 14.5±4 μg/mg protein (normal, n=6) even though there was a high variation in the level of monosaccharides among the SLE patients. An increase in oligosaccharides in α2‐macroglobulin from SLE patients compared to normal subjects was confirmed by concanavalin A (Con A) blots using peptide fragments derived from the micropurified protein. Since the interaction of peptide fragments derived from α2‐macroglobulin with Con A requires the presence of mannose and/or glucose residues, we have also examined if there are any correlations between the levels of mannose and glucose in α2‐macroglobulin and SLE. The concentration of mannose (38±60 μg/mg protein) in α2‐macroglobulin derived from SLE patients was significantly higher than normal donors (mannose, 4.8±1 μg/mg protein) however, the concentration of glucose in α2‐macroglobulin derived from SLE patients when compared to normal donors was not statistically significant, 18±20 μg/mg protein in SLE versus 2±0.5 μg/mg protein in normal donors due to high variation between samples. Also, the concentration of galactose in α2‐macroglobulin from SLE patients was significantly higher than normal donors (45.7±173 μg/mg protein versus 0.13±0.03 μg/mg protein). These results illustrate quantification of carbohydrate in selected glycoproteins such as α2‐macroglobulin may be a novel and alternative clinical marker for SLE.
Identification of a transcript predicting an alternative form of sulfated glycoprotein‐2 (clusterin) in rat tissues
Authors: Izawa Masao
Published: July 2007

As a clue to understand the role of androgen in rat seminal vesicle, we have isolated several cDNA clones of androgen‐responsive mRNAs. During further characterizations of these cDNAs were in progress, a distinct cDNA clone designated p116 was isolated. The sequence study suggested the expression of a transcript predictding an alternative form of Sulfated Glycoprotein‐2 (SGP‐2)/clusterin. RT‐PCR demonstrated the androgen‐dependent expression of p116 transcript in the seminal vesicles. The transcript was also detectable in the ventral prostate, the liver and the thymus. Since SGP‐2 has been suggested as a classical molecular marker of apoptosis, p116 transcript newly identified in the present study might provide a useful probe to further understand the roles of SGP‐2 and its related proteins during the course of apoptotic process not only in male accessory sex organs, but also in many types of cells undergoing apoptosis.
Reverse transcription‐polymerase chain reaction (RT‐PCR) for direct detection of cucumber mosaic virus (CMV) in gladiolus
Authors: Raj S. K. , Saxena Sangeeta , Hallan Vipin , Singh B. P.
Published: July 2007

A diagnostic method based on Reverse Transcription ‐ Polymerase Chain Reaction (RT‐PCR) was performed for detection of CMV genome directly in crude extracts of both healthy and infected gladiolus tissue using primers from the conserved sequences of CMV RNA‐3. RT‐PCR resulted in the amplification of 540 bp long fragment of CMV coat protein gene (CMV‐CP), as expected in most of the plant parts of infected gladiolus samples. Positive signals in Southern hybridization of amplified fragments with cloned CMV‐CP cDNA probe also confirmed the presence of CMV genome in gladiolus.
The stimulation of the mitochondrial permeability transition by dihydrolipoate and α‐lipoate
Authors: Saris Nils‐Erik L. , Karjalainen Ari , Teplova Vera V. , Lindros Kai O.
Published: July 2007

The effect of α‐lipoate and dihydrolipoate on the mitochondrial permeability transition was investigated. Both substances promoted the permeability transition in isolated rat liver mitochondria and in permeabilized hepatocytes, dihydrolipoate most potently in spite of it being a dithiol. The stimulation was prevented by Cyclosporin A or hydroxybutyltoluene but not by ascorbate. It is suggested that the greater potency of dihydrolipoate could be due to formation of a radical.
In vivo inhibition of Walker 256 tumour carnitine palmitoyltransferase I by soya oil dietary supplementation
Authors: Colquhoun Alison , de Mello Fábio E. P. , Curi Rui
Published: July 2007

The role of dietary fatty acids in the regulation of carnitine palmitoyltransferase (CPT) activity has been shown in liver but their role in the regulation of tumour CPT activity in vivo is unknown. The present study investigated the effects of several oils, given as dietary supplements, upon the activity of CPT I and II in the Walker 256 rat tumour and the inhibition or stimulation of tumour growth. CPT I activity was markedly inhibited by soya oil, rich in linoleic acid (70% inhibition vs control). CPT I mRNA expression was not inhibited by any of the oils studied, indeed soya oil caused a marked increase (132% vs control) in expression. These results suggest that soya oil can modulate, in vivo, the β‐oxidative pathway of tumour tissue and further supports the hypothesis of tumour CPT I regulation by polyunsaturated fatty acids.
Transition protein 1 from boar late spermatid nuclei having DNA‐melting activity is a dimeric protein
Authors: Akama Kuniko , Sato Hiroki , Hasegawa Shigeki , Shimada Ichio , Nakano Minoru
Published: July 2007

Polyacrylamide gel electrophoretic behavior of boar transition protein 1, TP1, under dissociating and non‐dissociating buffer conditions, and titration of fluorescently labeled TP1 with increasing amounts of TP1 showed that TP1 formed a dimer without intermolecular disulfide bond. TP1 dimer with intermolecular disulfide bond had similar DNA‐melting activity to TP1, but was not detected in extracts from boar late spermatid nuclei. These results suggest that TP1 dimer without intermolecular disulfide bond induces local destabilization of DNA in the late spermatid nuclei.
Univalent cation fluxes in yeast
Authors: Lapathitis Georgios , Kotyk Arnost
Published: July 2007

Transport of H+, K+, Rb+ and Tl+ ions was studied in a wild‐type strain of Saccharomyces cerevisiae and in its mutants defective in the high‐affinity K+ transport system TRK1 and in the double mutant with an additional deletion in the TRK2 gene. In the absence of glucose K+, Rb+ and Tl+ elicited a more or less stoichiometric exchange outflow of H+, in the mutants K+ moved out of cells even in the presence of 10 mM KCl or KNO3. In the presence of glucose in the wild type, K+, Rb+ and Tl+ brought about a massive outflow of H+ while being transported inward against high concentration gradients. In the trklΔ mutant the exchange fluxes were reduced by 65‐85%, in the double mutant those of K+, Rb+ and Tl+ practically cease but outflow of H+ caused by Tl+ remained at the level of the trklΔ mutant. It appears that, in addition to the H+ export by the PMAl‐coded plasma membrane H+‐ATPase, at least three different univalent‐cation involving activities are present: the high‐affinity transport system for K+ (TRK1), another system (possibly TRK2) with different responses to K+ and Rb+, vs. Tl+, and an active system for K+ export. The first two are apparently active exchange systems for K+, Rb+, and Tl+ against H+. The source of energy for these highly active transports (acting against gradients of 1000:1 and 5000:1, respectively) is unclear.
Studies on the heterologous expression of BstVI restriction endonuclease in Escherichia coli
Authors: Saavedra Claudia , González Enrique , Vásquez Claudio
Published: July 2007

Bacterial restriction and modification systems must be regulated to avoid self‐restriction. It is generally accepted that cognate DNA methyltransferases normally protects both, the host's chromosome and extrachromosomal elements from the activity of their endonuclease counterparts. When the bstVIRM genes from Bacillus stearothermophilusV were subcloned into Escherichia coli, several clones exhibiting a r+m‐ phenotype were originated. The present work was undertaken to analyze the possibility that mechanisms other than DNA methylation could account for the viability of these cells. No evidence was found for an inhibitory agent or endonuclease compartmentation. In vivo experiments showed that λ phage multiplication was poorly restricted by the heterologous enzyme. The restricting activity against the incoming phage increased however when phage adsortion was performed at higher temperatures. Analogous experiments in which a DNA‐repair deficient strain was used as a host for the thermophilic R‐M system suggested, to some extent, the participation of the repair machinery in the viability of r+m‐ clones.
A novel p53 mutation hotspot at codon 132 (AAG‐‐‐‐> AGG) in human renal cancer
Authors: Dahiya Rajvir , Deng Guoren , Selph Catherine , Carroll Peter , Presti Joseph
Published: July 2007

Mutations of p53 tumor suppressor gene are the most common genetic alterations in a variety of human carcinomas. The sites of p53 mutations, however, vary in different cancers. The present study was designed to characterize p53 mutations in 40 primary human renal cancer specimens using hot‐start‐PCR‐single‐strand conformation polymorphism (SSCP) analysis, sequencing of PCR product and immunohistochemistry. DNA extracted from microdissected paraffin‐embedded sections was amplified by hot‐start‐PCR using oligonucleotide primers specific for exons 4‐9 of p53. The mutations were analyzed by PCR‐SSCP technique and the generated fragments were denatured and analyzed by 6% polyacrylamide gel electrophoresis. The samples showing a band shift were denatured and sequenced using the SequenaseTM Version 2.0 DNA Sequencing Kit (US Biochemical, Cleveland, Ohio). Genomic DNA from control samples containing wild‐type p53 alleles was sequenced in parallel for confirming mutations in samples that were positive for p53 in the PCR‐SSCP analysis. The results of these experiments demonstrate that: (1) there were mutations in p53 exon 5 and 8 in 35% (14 out of 40 samples) of human renal cancer tissues as revealed by PCR‐SSCP analysis; (2) DNA sequencing of samples showing frame‐shift have hot spot of p53 mutation on exon 8 at codon 244 (GGC‐‐‐> TGC) and exon 5 at codon 132 [AAG(Lys)‐‐‐> AGG(Arg)]. This mutation in p53 exon 5 at codon 132 is novel and has not yet been reported; (3) immunohistochemical staining of p53 in renal cancer tissue using mouse anti‐human p53 monoclonal antibody, clone PAb 1801, correlated with the p53 mutation assessed by PCR‐SSCP. No correlation was found between p53 mutations and tumor stage and grade of renal cancer.
Influence of polyethylene glycol on lactate dehydrogenase
Authors: Lushchak Volodymyr I.
Published: July 2007

It is well established that polyethylene glycol (PEG) shifts the equilibrium in oligomeric protein systems to form higher molecular weight associates. We used this effect of PEG to evaluate a modification of functional properties of LDH from pig skeletal muscles. PEG decreases the rate of heating‐induced LDH inactivation in the concentration dependent manner. Michaelis constant and maximal velocity of the enzyme as well as inhibition of LDH by high pyruvate concentrations were affected by PEG. Enzyme preincubaion with PEG suppresses also the formation of ternary inactive complex NAD‐pyruvate‐LDH.
Detection of transmembrane helical segments at the nucleotide level in ukarayotic membrane protein genes
Authors: Lesnik T. , Reiss C.
Published: July 2007

The analysis of base distributions at the three codon positions, in sequences coding for polytopic membrane proteins from Eukaryotes, reveals a global excess of thymine and a depletion of adenine, at the second codon position. These genes were scanned using a sliding window, in which the average ratio of T over A at the second position of codons was computed. The scan shows that sharp peaks of this ratio, which are responsible for the high mean value of this parameter in the genes, correlate closely with the transmembrane segments of the membrane proteins. These results are quite similar to our previous findings for bacterial polytopic inner membrane proteins. This establishes the ratio of T over A at codon position two as a universal parameter for both the characterization of the genes of polytopic membrane proteins, and for the location of alpha‐helical, transmembrane segments of these proteins.
Inosine‐5′‐monophosphate analogues as inhibitors of human IMP cyclohydrolase and cellular growth
Authors: Szabados Eve , Manthey Michael K. , Wilson Paul K. , Christopherson Richard I.
Published: July 2007

The catalytic mechanism for the enzyme, IMP cyclohydrolase, may involve a reaction intermediate with negative charge in the 2‐position of the purine ring (Szabados, E., Hindmarsh, E., Phillips, L., Duggleby, R.G. & Christopherson, R.I. (1994) Biochemistry 33, 14237‐14245). Threeanalogues of IMP have been synthesised where fluorine, chlorine or bromine has been substituted in the 2‐position on the purine ring. These analogues with an electronegative substituent may resemble a reaction intermediate for IMP cyclohydrolase; 2‐fluoro IMP is a potent inhibitor of the enzyme with a Ki value of 0.19 μM, while 2‐chtoro IMP has a Ki of 1.9 μM and 2‐bromo IMP is not inhibitory. However, IMP cyclohydrolase is not inhibited in human CCRF‐CEM leukaemia cells exposed to 2‐fluoro inosine although it is toxic to these cells with an IC50 value of 4.9 μM.
The effect of ribosome‐inactivating proteins on the ribosome from the hyperthermophilic archaeon Sulfolobus solfataricus
Authors: Raimo Gennaro , Arcucci Alessandro , Masullo Mariorosario , Bocchini Vincenzo , Barbieri Luigi , Valbonesi Paola , Stirpe Fiorenzo
Published: July 2007

Protein synthesis in the thermoacidophilic archaeon Sulfolobus solfataricus (Ss) was inhibited by polynucleotide:adenosine glycosylase activity of some type 1 ribosome‐inactivating proteins (RIP). The target of RIP was S. solfataricus rRNA that was depurinated thus producing inactive ribosomes. The amount of RIP required to half‐inactivated Ss‐ribosomes was comparable to that needed for eubacterial ribosomes, but two orders of magnitude higher than that required for mammalian ribosomes. In addition, RIP treated Ss‐ribosomes were also less efficient in stimulating the ribosome dependent GTPase activity of the S. solfataricus elongation factor 2 (SsEF‐2) thus suggesting that the inhibition of protein synthesis was probably due to the lack of the interaction between depurinated Ss‐ribosomes and SsEF‐2. Since SsEF‐2 protects Ss‐ribosomes against RIP activity it can be hypothesised that also on Ss‐ribosomes the sites of interaction for the translocation factor 2 and the RIP are topographically close.
Role of naturally occuring autoantibodies in senescence of normal and ATP depleted goat erythrocytes
Authors: Anwar Adil , Saleemuddin Mohammed
Published: July 2007

The possible role of autoantibodies in the senescence of goat erythrocytes has been investigated. For this purpose goat gammaglobulin was purified to homogenity and antigoat gammaglobulin was raised in rabbits. Using the peroxidase labelled antigoat gammaglobulin it was possible to detect the presence of auto anti band‐3 antibodies in goat sera. The goat erythrocytes were aged in vitro by ATP‐depletion, which resulted in appearence of a 52,000 mol. wt. polypeptide. The in vitro aged goat erythrocyte membrane bound substantial amounts of auto anti band‐3 antibodies as visualized by immumoblots.
Response to platelet‐derived growth factor by phenotypically different cultured human aortic smooth muscle cells
Authors: Kato Masahiro , Morimatsu Minoru , Tanimoto Akihide , Arima Nobuyuki , Sasaguri Yasuyuki
Published: July 2007

We investigated the effects of PDGF on DNA synthesis and mitogen‐activated protein (MAP) kinase activity, and demonstrated that the adult intimal SMC was concentration‐dependently stimulated by all PDGF isoforms in terms of both [3H]thymidine incorporation and MAP kinase activation, with PDGF‐BB and ‐AB being more potent than PDGF‐AA. The intimal SMCs and the neonatal SMCs showed a similar response with regard to MAP kinase activation. On the other hand, the intimal SMCs expressed many more PDGF receptors than the adult medial SMCs, which expressed a greater amount of PDGF‐A chain mRNA and showed a lesser response to PDGFs. These results suggest that the intimal SMCs have a relatively high potential to react to exogenous PDGFs, whereas the adult medial SMCs depend on endogenous or autocrine secretion of PDGF‐AA.
Effects on larvicidal activity of single proline substitutions in α3 or α4 of the Bacillus thuringiensis Cry4B toxin
Authors: Uawithya Panapat , Tuntitippawan Tipparat , Katzenmeier Gerd , Angsuthanasombat Chanan , Panyim Sakol
Published: July 2007

The possible role of α‐helices 3 and 4 in toxicity of the dipteran‐active Bacillus thuringiensis Cry4B δ‐endotoxin was investigated by employing proline substitutions via site‐directed mutagenesis. Similar to the wild‐type Cry4B, the mutant toxins were over‐expressed in Escherichia coli as cytoplasmic inclusions and were structurally stable upon solubilization and trypsin activation. The substitution of glutamine 149 by proline in the center of helix 4 (Q149P) resulted in a nearly complete loss of toxicity against Aedes aegypti mosquito‐larvae. However, single proline replacements near the center of helix 3 (V119P) and at the N‐terminus of helix 4 (Q140P) did not decrease larvicidal activity. The toxicity of E. coli cells expressing the wild‐type toxin was significantly reduced by two‐hour preincubation with the non‐toxic mutant (Q149P), thus indicating that the primary binding step was not affected by the proline substitution in helix 4. The results therefore reveal a crucial role for helix 4 of the Cry4B toxin in toxicity, possibly in membrane insertion and pore formation rather than in receptor recognition.
Identification of new GATA factor family genes in Saccharomyces cerevisiae and Schizosaccharomyces pombe
Authors: Hoe Kwang‐Lae , Chung Kyung‐Sook , Won Misun , Yoo Ook‐Joon , Yoo Hyang‐Sook
Published: July 2007

By genomic Southern hybridization using degenerate oligo probes corresponding to the conserved zinc finger of the GATA family, four and three new GATA‐factor genes were detected in Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. Through degenerate PCR and Southern hybridization using an internal probe between the PCR primers, a new GATA‐factor gene of S. pombe, gafl, was identified. The DNA sequence analysis revealed that the partial gafl gene contains the strictly conserved zinc finger which is the hallmark of the GATA family. As judged form the codon usage of leucine in the zinc finger probe, the zinc fingers of S. pombe are more similar to those of higher fungi than those of S. cerevisiae, and this means that S. pombe is phylogenically close to higher organisms than S. cerevisiae.
V1a receptor mRNA expression in mouse early proximal tubule (S1) cell line is regulated by protein kinase A
Authors: Takeda Michio , Kobayashi Mami , Shirato Isao , Endou Hitoshi
Published: July 2007

The purpose of this study was to investigate the regulation of V1a receptor gene expression in mouse early proximal tubule (S1) cell line (NF‐5). NF‐5 cells showed arginine vasopressin (AVP)‐induced increase in cytosolic free calcium concentration mediated by V1a receptor. Polymerase chain reaction coupled with reverse transcription analysis revealed that forskolin, but not phorbol‐12‐myristate‐13‐acetate stimulated V1a receptor mRNA expression in NF‐5 cells. Our data suggest that V1a receptor gene expression is regulated by protein kinase A in proximal tubule cell line, and the cell line might be a useful model for the further studies of the function and the regulation of the activity and expression of the V1a receptor in the proximal tubule.
Spectroscopic studies of the interactions of the pyrethroid insecticide fenvalerate with gramicidin
Authors: Ghosh J. K. , Sarkar S. N. , Sikdar S. K.
Published: July 2007

Fenvalerate is a pyrethroid insecticide which interacts with ionic channels. Using circular dichroism technique we have studied the interaction of fenvalerate with gramicidin, a model channel peptide which transports ions. In most organic solvents, gramicidin exists as a double helix except in trifluoroethanol where it exists as a channel forming single stranded β6.3 helical monomer. In model lipid membranes, under certain experimental conditions, gramicidin exists as a channel forming single stranded β6.3 helical dimer. Our results show that fenvalerate interacts more with the single stranded β6.3 helical monomer or dimer than with the double helical form of gramicidin. This was further confirmed by an increase in the rate of gramicidin mediated proton transport in liposomes by fenvalerate, using the pH sensitive fluorophore, pyranine.
Variations in gene expression and genomic stability of human hepatoma cells integrated with hepatitis B virus DNA
Authors: Kuo Kou‐Wha , Yang Pei‐Yi , Huang Yu‐Sheng , Shieh Dar‐Zen
Published: July 2007

The association of hepatitis B virus (HBV) infection with human hepatoma is well established. However, no consensus regarding the etiology of hepatocellular carcinoma was elucidated. In this paper, the genomic stability and gene expression of HBV DNA‐integrated and non‐integrated hepatoma cells by Transcript Profile (TP)‐PCR and RT‐PCR are characterized. The additional DNA bands generated from TP‐PCR of HBV integrated genomes were not correlated with the sequence of HBV, suggesting that the variations may result from genomic instability of the host cells. Moreover, differential genes expressed in HBV DNA‐integrated cells were sequenced. A cDNA generated from the integrated cells exhibited 99.3% homology with the sequences of ATP synthase 6 and cytochrome C oxidase III, but the sequences were abnormally linked together. Since HBV infection may alter the energy metabolism of the cell, the results suggest that the integration may cause mitochondriae defects in the ATP synthase 6 and cytochrome C oxidase III genes.
Effects of short‐ and long‐term exposure to linoleic acid hydroperoxide on cytosolic calcium ion level of human aortic intimal smooth muscle cells In vitro
Authors: Arima Nobuyuki , Sasaguri Yasuyuki , Yagi Kunio
Published: July 2007

When cultured intimal smooth muscle cells from human aorta were exposed for a short period of time (25 sec) to linoleic acid hydroperoxide (100 nmol/ml), influx of calcium ions into the cytosol of these cells was provoked, and a temporal increase in cytosolic calcium ion level was observed. Two calcium channel blockers inhibited this influx. Long‐term exposure (4 min) of these cells to the hydroperoxide also provoked the influx of calcium ions, which resulted in a longer time of the calcium ion increase. In the latter case, however, the calcium channel blocker inhibited the initial influx, but then the influx started and continued even in the presence of the blocker. Such difference in exposure time‐dependent effects should be taken into account in considering pathological roles of lipid hydroperoxides.
Effects of ethanol ingestion on insulin binding to rat leydig cells
Authors: Srikanth V. , Shanthakumari D. , Sudha S. , Govindarajulu P. , Balasubramanian K.
Published: July 2007

Effects of ethanol treatment and its withdrawal on insulin binding to isolated rat Leydig cells were Studied. Mature rats were given ethanol by gastric intubation for 30 days at a dose of 3.0g/kg body weight, twice daily, as a 25% (v/v) aqueous solution and treatment was withdrawn for the subsequent 30 days in an another group. Ethanol treatment markedly increased serum insulin and reduced the 125I‐insulin binding to Leydig cells and the activities of Leydig cellular steroidogenic enzymes such as 3β‐HSD and 17β‐HSD. Withdrawal of ethanol treatment restored these changed values to their normal levels. The results suggest the possible involvement of subnormal insulin actions, as that of LH, in the ethanol‐induced impairment of Leydig cellular steroidogenesis and the resulting hypoandrogenization associated with alcohol abuse.
The inhibitory effect of benzodiazepine derivatives on the bovine lens aldose reductase enzyme
Authors: Evcimen Net Das , Pekiner Bilgehan , Süzen Sibel , Büyübingöl Erdem
Published: July 2007

The aldose reductase enzyme, involved in the sorbitol pathway which is an important mechanism in regulation of mammalian glucose metabolism, has been known to play a significant role in the initiation of diabetic complications. Numerous chemical substances have been prepared in order to improve the pharmacological profile of inhibition of aldose reductase enzyme. In this study, aldose reductase inhibitory activities of several benzodiazepine derivatives were investigated. The enzyme was obtained from bovine lenses via the ammonium sulphate‐protein out method with several steps. It was found that tetrazepam had a significant inhibitor potency among the other benzodiazepine derivatives showing very slight inhibitor activities that are indicated in terms of percent inhibitor potency at 10‐4 M concentration.
tRNA aminoacylated at high pressure is a correct substrate for protein biosynthesis
Authors: Krzyzaniak Andrzej , Twardowski Tomasz , Barciszewski Jan , Sałanski Piotr , Jurczak Janusz
Published: July 2007

tRNA can be aminoacylated specifically with amino acids at high pressure of 6 kbar (1 bar = 1.013 atm = 0.1 MPa = 105 Pa) in the absence of the specific aminoacyl‐tRNA synthetase and ATP. In this paper we present new evidence obtained by HPLC chromatography and TLC analysis that the esterification reaction under pressure really takes place at the 3′ end of the tRNA molecule. If so, tRNA to be aminoacylated undergoes conformational changes similar to those induced with aminoacyl‐tRNA synthetase. The most important finding is that aminoacyl‐tRNA obtained at high pressure binds to ribosomes and participates in the synthesis of polyphenylalanine in vitro. This is the best proof of proper charging of tRNA at high pressure.
Characterization of a novel DNA binding domain within the amino‐terminal region of the RAG‐1 protein
Authors: Alva Jackelyn A. , Lin Albert , Lyon Christopher J. , Aguilera Renato J. , Galic Zoran
Published: July 2007

Rag‐1 and Rag‐2 are the critical components of the V‐(D)‐J recombinase required for site‐specific recombination of the antigen receptor genes. In this study, we have examined the ability of recombinant (r) Rag‐1 and Rag‐2 to bind the recombination signal sequences (RSS) and have determined that rRag‐1, but not rRag‐2, is able to directly bind DNA. rRAG‐1 DNA binding activity was found to reside within a novel amino‐terminal arginine‐rich (RR) domain with partial homology to a variety of nucleic acid binding domains. Although the RR‐domain did not demonstrate RSS‐specificity, this DNA binding domain may stabilize the interaction of RAG‐1 with, or increase the affinity for, the V‐(D)‐J recombination signals.
Changes of isoenzymes of serum N‐acetyl‐β‐D‐glucosaminidase in relation to different types of diabetes
Authors: Mandic Ljuba , Filipovic Dragana
Published: July 2007

The activity of the isoenzymes of N‐acetyl‐β‐D‐glucosaminidase (NAG, EC 3.2.1.30) is determined in the serum of insulin‐dependent (IDDM) and non‐insulin‐dependent diabetics (NIDDM) with or without diabetic complications. The increase in total activity of serum NAG in diabetics is proportional to the A form activity (r=0,976, p< 0,0001). The contribution of the A form activity (65.87±5.99%) to total NAG activity of IDDM and NIDDM diabetics with and without complications does not change considerably compared to the control group. The contribution of the B form activity depends on the state of metabolic monitoring and diabetic complications. A significantly lower activity of the serum B form was found in IDDM (p<0,001) and NIDDM diabetics (p<0,05) compared to the healthy individuals, as well as the higher activity ratios of the A/B forms. A decrease in serum B form is correlated with the occurrence and abundance of the intermediary I forms (r=‐0,665). These changes are particularly significant in the individuals with the pronounced microangiopathy.
Tata element‐binding protein is important to epidermal growth factor‐dependent induction of H2B histone gene expression in primary hepatocytes from rat
Authors: Lim Kyu , Song Hea‐Jeong , Yun Kyung‐Ah , Son Mee‐Young , Park Jong‐Il , Kweon Gi‐Ryang , Hwang Byung‐Doo , Byun Sang‐Hyun , Yoon Wan‐Hee
Published: July 2007

Epidermal growth factor (EGF) is a potent mitogen for rat hepatocytes and mammalian histone synthesis is functionally and temporally coupled to DNA replication. To gain an insight on the role of EGF in the regulation of H2B histone gene expression in primary hepatocyte cultures, the binding patterns of nuclear proteins to various elements in the H2B histone gene upstream region have been investigated. EGF induced H2B histone mRNA with maximal stimulation reached at 36 hours. The induction of H2B histone mRNA was dependent on the concentration of EGF and almost reduced by actinomycin‐D pretreatment. In DNase I footprinting analysis, one nuclear factor (TATA element‐binding protein, TBP) bound at ‐20 bp (TATA element)in either the absence or presence of EGF. One DNA‐protein complex was formed by DNA mobility shift assay when TATA element was incubated with nuclear extract prepared from EGF‐free hepatocytes, and the amount of TBP was increased after EGF treatment. These results suggest that TBP may be correlated with transcriptional regulation of H2B histone gene by EGF in primary hepatocytes.
Development of a competitive enzyme linked immuno sorbent assay to measure ceruloplasmin in γ‐irradiated rat serum
Authors: Kim In Gyu , Park Seon Young , Kim Kug Chan
Published: July 2007

The concentrations of ceruloplasmin could not be determined by comparing the oxidase activities toward p‐phenylenediamine and o‐dianisidine in rat whole serum with those of purified ceruloplasmin because of the different specific amine oxidase activities of ceruloplasmin purified from normal and γ‐irradiated rats and the lability caused by freezing and thawing. A competitive enzyme linked immunosorbent assay with antigens immobilized on the solid phase was developed to measure the ceruloplasmin in rat serum. The blocking materials and pH of the coating buffer had an effect on the amount of ceruloplasmin bound to the microtiter plates. The blocking materials nonspecifically reacted with the rabbit anti‐rat ceruloplasmin IgG, and consequently the coating sensitivity was decreased and standard curves were not formed completely. Because rabbit anti‐rat ceruloplasmin IgG did not show any cross reactivity with rat albumin and hemoglobin, these proteins did not affect the assay at a concentration of 200 μg/ml. This assay is useful for measuring the concentration of ceruloplasmin in normal and irradiated rat serum.
A convenient method of DNA extraction from blood anticoagulated wity EDTA
Authors: Yokota M. , Sindo K. , Hiyoshi M. , Tsuda I. , Tatsumi N.
Published: July 2007

Surplus blood often remains after routine clinical tests using EDTA‐anticoagulated samples. To use this blood for DNA analysis, we isolated white cells by adherence to polyethylene terephthalate fibers, which could be stored for several weeks transported by mail after methanol fixation. DNA yield was sufficient and correlated with white cell count. Extracted DNA was free of hemoglobin contamination and durable to polymerase chain reaction and enzyme digestion, which yielded products visualized as well‐separated bands on electrophoresis. We found our method to be practical for the routine clinical laboratory.
Amplification of phenylalanine hydroxylase and cystathionine β‐synthase transcripts in human peripheral lymphocytes by RT‐PCR
Authors: Devi K. Santhi , Devi A. Radha Rama , Kondaiah Paturu
Published: July 2007

A simple, rapid, reliable and convenient method was developed to analyze the gene defects in Phenylketonuria (PKU) and Homocystinuria (HCU). In this method, illegitimately transcribed phenylalanine hydroxylase (PAH) and cystathionine β‐synthase (CBS) mRNAs in peripheral lymphocytes were used as templates for amplification by RT‐PCR. The amplified products were confirmed by restriction enzyme digestions, southern blot hybridizations and sequencing. Point mutations in the protein coding region and splice junction mutations of PAH and CBS can be analyzed by this method.
De novo protein synthesis is essential for thermotolerance acquisition in a Saccharomyces cerevisiae trehalose synthase mutant
Authors: Gross Claudia , Watson Kenneth
Published: July 2007

Heat shock (25°C to 37°C for 30 min) acquisition of thennotolerance (at 50°C) was observed in a yeast trehalose synthase mutant and the corresponding control strain. The acquisition of thermotolerance in the control strain was maintained for a significantly longer time in the trehalose synthase mutant. The heat shock was associated with the synthesis of specific heat shock proteins and, in the case of the control strain, also trehalose accumulation. Inhibition of protein synthesis during the heat shock totally abolished acquisition of thermotolerance in both strains but not trehalose accumulation in the control. It was concluded that trehalose may only be required for prolonged stress protection while heat shock proteins are required for heat shock acquisition of thermotolerance.
DNA‐binding properties of the overexpressed recombinant estrogen receptor α
Authors: Ikeda Mitsunori , Tsuji Norio , Kikukawa Kyoko , Asahar Yoshie , Nakashima Aya , Minatogawa Yohsuke
Published: July 2007

Estrogen receptor α was overexpressed in COS‐7 cells by transformation of an expression vector with the full length open reading frame of the receptor α. The nuclear estradiol‐receptor complex and the soluble receptor from the COS‐7 cells were cross‐linked with an estrogen response element (ERE), which was substituted with 5‐bromo‐2′‐deoxyuridine (BrdUVRE), as the receptor dimers by UV irradiation. In gel retardation analysis, the specific bindings of both nuclear and soluble receptors to ERE were decreased with increasing of KCl concentration compared with 0.1 M KCl. The ionic interactions of both receptors with ERE are thought to be similar.
Cloning and expression analysis of a cDNA coding for a dexamethasone‐inducible cytochrome P450 in Xenopus laevis liver
Authors: Ohi Hiroaki , Sugata Eiji , Fujita Yoshiaki , Saito Hiromi , Saguchi Ken‐ichi , Murayama Nobuhiro , Higuchi Shigesada
Published: July 2007

We previously purified a cytochrome P450 (P450) from liver microsomes of adult female Xenopus laevis. In this study, we screened a cDNA library of Xenopus liver to isolate the cDNA clone coding for this P450. The 5′‐end of the resultant cDNA was truncated at the N‐terminal region and extended by method of rapid amplification of eDNA end to give the complete coding sequence. Amino acid sequence showed this clone to be 36% to 55% identical to members of the CYP2 family and less than 31% identical to members of other gene famines, and to belong to the CYP2Q subfamily. This gene is expressed constitutively in the livers of adult male and female frogs, and is significantly induced by dexamethasone administration.
Possible target components for the inhibitory effect of N‐ethylmaleimide on the activation of neutrophil NADPH oxidase
Authors: Park Jeen‐Woo , Park Hee‐Sae , Lee Su Min
Published: July 2007

Potential target components for the inhibitory effect of covalent sulfhydryl‐modifying reagent N‐ethylmaleimide (NEM) on the activation of NADPH oxidase in human neutrophils was studied in a cell‐free system. The capacity of both cytosol and membrane fractions to induce the translocation of cytosolic components and O2‐ generation in the cell‐free activation system was affected by NEM. The phosphorylation of p47phox, which mediates the translocation of cytosolic complex, by protein kinase C was not inhibited by NEM and NEM‐treated p47phox was as effective as untreated p47phox both in the kinase‐dependent and in the amphiphile‐dependent cell‐free activation sysyems. In addition, phosphatidic acid‐dependent phosphorylation of cytosol including p47phox was not affected by NEM. The inhibition of cytosol's capacity to activate NADPH oxidase was partially reversed by an addition of the fraction containing G‐protein rac. Taken together, the data suggest that membrane component cytochrome b558 and cytosolic component rac may be the potential targets for the NEM effect on the activation ofNADPH oxidase.
Streptokinase secretion by Serratia marcescens signaled by the C‐terminal 41 amino acid segment of metalloprotease
Authors: Kim Ki Seok , Bae Kwang Hee , Kim Il Chul , Byun Si Myung , Shin Yong Chul
Published: July 2007

In order to investigate the secretion signal of Serratia marcescens metalloprotease (SMP) and examine the ability of the secretion signal to secrete foreign proteins, hybrid genes encoding the passenger‐SMP C‐terminal segments were constructed. As a passenger protein, streptokinase (SK) deprived of its signal peptide was used. Three kinds of SMP C‐terminal segments containing 41, 80, or 220 amino acid residues were fused to the C‐terminus of SK as secretion signals. The SK‐SMP chimeric proteins containing 41 or 220 amino acid segments of the SMP C‐terminus were secreted into the culture medium by the SMP transporter of S. marcescens. This result suggests that cytoplasmic SK is secreted into the external medium by the C‐terminal segments of SMP and also shows that the smallest, 41 amino acid segment of the SMP C‐terminus functions as a secretion signal of foreign proteins as well as SMP.
Altered membrane skeleton of red blood cells participates in cadmium‐induced anemia
Authors: Hamada Tetsuo , Tanimoto Akihide , Arima Nobuyuki , Ide Yoshihiro , Sasaguri Takakazu , Shimajiri Shohei , Sasaguri Yasuyuki
Published: July 2007

Poikilocytosis of red blood cells (RBCs) was observed to be associated with anemia in rats given subcutaneous injections of cadmium (Cd). Phase‐contrast light and scanning electron microscopic examinations revealed that acanthocytes appeared in the early stages of administration, and that the number of RBC fragments increased later. Ultrastructural analysis of RBC ghosts by negative staining demonstrated that the normal lattice structure of the membrane skeleton was abolished. The osmotic fragility curve of the Cd‐exposed RBCs disclosed that most of the cells were less fragile than control RBCs. These data indicate that the RBC membrane skeleton is initially altered by Cd‐exposure, followed by deformation of the cell, thus promoting intrasplenic hemolysis, and resulting in anemia.
Neonatal hyperbilirubinemia and mutation of the bilirubin uridine diphosphate‐glucuronosyltransferase gene: a common missense mutation among Japanese, Koreans and Chinese
Authors: Akaba Kazuhiro , Kimura Toshiyuki , Sasaki Ayako , Tanabe Saori , Ikegami Tohru , Hashimoto Motoya , Umeda Hitoshi , Yoshida Hiroshi , Umetsu Kazuo , Chiba Hitoshi , Yuasa Isao , Hayasaka Kiyoshi
Published: July 2007

We analyzed the bilirubin uridine diphosphate‐glucuronosyltransferase (B‐UGT) gene in 42 Japanese newborns with hyperbilirubinemia and determined that 21 infants were heterozygous while 3 was homozygous for Gly71Arg. Allele frequency of Gly71Arg was 0.32 in newborns with hyperbilirubinemia, which was significantly higher than 0.13 in healthy Japanese controls. This mutant allele is also prevalent among Korean and Chinese healthy controls with a frequency of 0.23 in both populations. However, this mutation was not detected in 50 healthy German controls. These data suggest that the high frequency of the Gly71Arg mutation of the B‐UGT gene is associated with high incidence of neonatal hyperbilirubinemia in Japanese, Korean and Chinese populations.
EPC‐K1 attenuates peroxynitrite‐induced apoptosis in cerebellar granule cells
Authors: Wei Taotao , Chen Chang , Zhao Baolu , Xin Wenjuan , Mori Akitane
Published: July 2007

Apoptosis induced by peroxynitrite in cultured cerebellar granule cells was confirmed morphologically by chromatin condensation and biochemically by DNA laddering. A 30 min exposure to peroxynitrite (10 μM) initiated oxidative stress, which caused the formation of thiobarbituric acid‐reactive substances (TBARS) and the alteration of cell membrane fluidity. Peroxynitrite treatment also caused ATP decrease and thus activated the apoptotic program. Pre‐treating cells with antioxidant EPC‐K1 (L‐ascorbic acid 2‐[3,4‐dihydro‐2,5,7,8‐tetramethyl‐2‐(4,8,12‐trimethyltridecyl)‐2H‐1‐benzopyran‐6‐yl‐hydrogen phosphate] potassium salt), a new water‐soluble derivative of vitamin C and vitamin E, attenuated oxidative injury and prevents cells from apoptosis. The results suggest that EPC‐K1 might be used as a potential therapeutic agent for diseases associated with NO/ONOO‐‐mediated neuronal injury.
Insulin rapidly induces nuclear translocation of PI3‐Kinase in HepG2 cells
Authors: Kim Sung‐Jin
Published: July 2007

Insulin action on nuclear PI3‐Kinase and IRS‐1 was explored in HepG2 cells. Following insulin treatment, the cells were subjected to subcellular fractionation. Western blot analyses were carried out to identify IRS‐1 and PI3‐Kinase in the nuclear and postnuclear preparations. IRS‐1 protein was identified in the nucleus under basal condition. Insulin had no effect in the content of nuclear IRS‐1. In contrast, PI3‐Kinase was not detected under basal condition. However, insulin treatment for 1 to 10 min caused significant increase of PI3‐Kinase in the nucleus while it induced corresponding decrease of PI3‐Kinase in cytoplasm. Strikingly, Insulin stimulated the association of IRS‐1 and PI3‐Kinase in the nucleus in a similar kinetics with the nuclear translocation of PI3‐Kinase. These results suggest that insulin induces nuclear tranlocation of PI3‐Kinase and the translocated PI3‐Kinase associates with nuclear IRS‐1. The association of IRS‐1 and PI3‐Kinase in the nucleus in response to insulin may play important roles in nuclear insulin actions.
Oxygen free‐radicals mediate the damaging effect of ultraviolet light on membrane mitochondria
Authors: Chávez Edmundo , Zazueta Cecilia , Cuéllar Adela , Reyes‐Vivas Horacio , García Noemí
Published: July 2007

This paper reports evidence that exposure of mitochondria to near‐ultraviolet light inhibits electron transport, collapses the electric gradient, and increases non‐specific membrane permeability to matrix solutes such as Ca2+. Membrane energization, as well as superoxide dismutase and catalase avoid membrane leakiness. Increased permeability correlates with a diminution in the titrated thiol groups. Plausibly the pore is formed through the formation of sulfhydryl bridges by the action of UV light‐derived oxygen‐centered free‐ radicals on membrane proteins.
An essential lysine residue of green crab (Scylla Serrata) alkaline phosphatase
Authors: Chen Qing‐Xi , Zhou Hai‐Meng
Published: July 2007

The values of pKa (10.38) and Hion (10.92 Kcal/mol) have been determined for the ionizing groups controlling activity of green crab alkaline phosphatase. The results suggest that ‐NH2 of lysine residue responsible for the ionzation with pKc=10.38 and ΔH° is in the active site of the enzyme. Modification of lysine residues of the enzyme by an excess of 2,4,6‐trinitrobenzenesulfonic acid leads to complete inactivation. The two results coincide with each other. Quantitative assessment of the data indicates that among the reactive ‐NH2 groups modified only one is essential for the activity of the enzyme.
The DNA sequence specificity of hedamycin damage determined by ligation‐mediated PCR and linear amplification
Authors: Cairns Murray J. , Murray Vincent
Published: July 2007

The DNA sequence specificity of hedamycin damage was compared using two techniques: Ligation‐mediated PCR (LMPCR) and Linear Amplification (LA). The electrophoretic mobility of LA products were made comparable with LMPCR products by using a primer with a 27 bp non‐binding 5′‐extension. By direct comparison of the damage intensity (as represented by each of the methods), considerable bias was found in the LMPCR system with respect to LA‐resulting in the under representation of lesions with T as the base 3′ to the damage site. In view of this observation some caution should be exercised in the interpretation of data for DNA damage/repair specificity derived by LMPCR. In addition the extended primer LA method used in these experiments, could be applied to generate a dideoxy sequencing ladder for analysis of LMPCR products. This would negate the need to prepare Maxam‐Gilbert chemical sequencing fragments for amplification through LMPCR.
Increased metabolism of peroxynitrite‐modified [125I] β‐VLDL by monocytes from hypercholesterolemic rabbits
Authors: Silva E.L. da , Abdalla D. S. P.
Published: July 2007

The metabolism (uptake and degradation) of peroxynitrite‐modified β‐VLDL and native β‐VLDL labeled with [125I]iodine was studied in monocytes isolated from peripheral blood of hypercholesterolemic (HC) and normolipidemic rabbits (NC). The peroxynitrite‐modified β‐VLDL uptake and degradation was up to 2‐fold higher in monocytes from HC rabbits than monocytes from NC rabbits. In addition, monocytes from HC rabbits took up and degraded significantly more peroxynitrite‐modified β‐VLDL than native β‐VLDL. In contrast, monocytes from NC rabbits took up both lipoproteins in a similar rate and degraded more native β‐VLDL than peroxynitrite‐modified β‐VLDL. Our results suggest that hypercholesterolemia can affect monocyte metabolism enhancing foam cell formation by increasing the influx of peroxynitrite‐modified β‐VLDL.
Changes in the activities of mitochondrial enzymes in the progress of tumorigenesis of bladder cancer
Authors: Huang Chun‐Nung , Tsai Jin‐Lian , Chen Ming‐Tan , Wu Wen‐Jeng , Kuo Kou‐Wha , Huang Chun‐Hsiung
Published: July 2007

To investigate changes in the activities of mitochondrial enzymes in the progress of tumorigenesis of bladder cancer, N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine (BBN) carcinogen was administered orally to male Sprague‐Dawley rats for eight weeks. The urinary bladders were harvested periodically for the evaluation of tumorigenesis. The activities of NADH cytochrome c reductase (NCCR), succinate cytochrone c reductase (SCCR) and cytochrome c oxidase (CCO) were measured. The NCCR and SCCR activities elevated significantly by the exposure of BBN and decreased rapidly when BBN was withdrawn. However, the CCO activity increased and reached plateau at 18 weeks in spite of the discontinuance of BBN. The results indicated that the NCCR, SCCR and CCO activities were significantly elevated in the tumorigenesis. However, the CCO enzyme may be more related to the progress of tumorigenesis of bladder cancer.
The interfacial behavior of cytochrome c studied by pendant‐drop technique
Authors: Gazová Zuzana , Bágelová Jaroslava , Antalík Marián , Tomori Zoltán
Published: July 2007

The adsorption properties of cytochrome c (cyt c) were characterized by surface tension measurements using the pendant‐drop method employing the digital image analysis of the drop shape. The method was applied to the study of the protein conformation change due to acidification at low ionic strength. The observation of the saturated steady‐state surface tension shows that decrease in pH induces its cooperative change centered around pH 2.5. This value is equal to the value of apparent pK of the acid‐induced transition of the horse ferricyt c from a native state to the unfolded conformation. This indicates that the saturated steady‐state surface tension is sensitive to the conformation of cyt c in bulk phase, and the pendant‐drop method might be used to monitor changes in the tertiary structure of proteins.