bluuebunny/crossref_metadata_2025 huggingface dataset:
Potassium Channels in the Peripheral Microcirculation
Authors: JACKSON WILLIAM F.
Published: July 2007

Vascular smooth muscle (VSM) cells, endothelial cells (EC), and pericytes that form the walls of vessels in the microcirculation express a diverse array of ion channels that play an important role in the function of these cells and the microcirculation in both health and disease. This brief review focuses on the K+ channels expressed in smooth muscle and endothelial cells in arterioles. Microvascular VSM cells express at least four different classes of K+ channels, including inward‐rectifier K+ channels (KIR), ATP‐sensitive K+ channels (KATP), voltage‐gated K+ channels (KV), and large conductance Ca2 +‐activated K+ channels (BKCa). VSM KIR participate in dilation induced by elevated extracellular K+ and may also be activated by C‐type natriuretic peptide, a putative endothelium‐derived hyperpolarizing factor (EDHF). Vasodilators acting through cAMP or cGMP signaling pathways in VSM may open KATP, KV, and BKCa, causing membrane hyperpolarization and vasodilation. VSM BKCa may also be activated by epoxides of arachidonic acid (EETs) identified as EDHF in some systems. Conversely, vasoconstrictors may close KATP, KV, and BKCa through protein kinase C, Rho‐kinase, or c‐Src pathways and contribute to VSM depolarization and vasoconstriction. At the same time KV and BKCa act in a negative feedback manner to limit depolarization and prevent vasospasm. Microvascular EC express at least 5 classes of K+ channels, including small (sKCa) and intermediate (IKCa) conductance Ca2 +‐activated K+ channels, KIR, KATP, and KV. Both sK and IK are opened by endothelium‐dependent vasodilators that increase EC intracellular Ca2 + to cause membrane hyperpolarization that may be conducted through myoendothelial gap junctions to hyperpolarize and relax arteriolar VSM. KIR may serve to amplify sKCa‐ and IKCa‐induced hyperpolarization and allow active transmission of hyperpolarization along EC through gap junctions. EC KIR channels may also be opened by elevated extracellular K+ and participate in K+‐induced vasodilation. EC KATP channels may be activated by vasodilators as in VSM. KV channels may provide a negative feedback mechanism to limit depolarization in some endothelial cells.
Survival of the weakest: the differential improvement of schools causing concern in England
Authors: Matthews Peter , Sammons Pam
Published: July 2007

Inspection and performance data show that the schools identified as least effective in England (in special measures) are more likely to sustain the improvement they make after inspection than those that are relatively more effective, although still causing concern (identified as having
serious weaknesses). We compare the progress of these two categories of schools causing concern and discuss the role of school leadership in relation to their differential improvement trends. It is argued that special measures identification and support for improvement has made an important
contribution to the policy goals of promoting educational inclusion and raising standards.
Effect of IL‐1 in amniotic fluid on the fetal keratinocyte
Authors: Kamada Masaharu , Hirano Kohki , Arase Seiji , Daitoh Toshifumi , Maegawa Masahiko , Irahara Minoru , Aono Toshihiro
Published: July 2007

IL‐1 α is predominantly increased in amniotic fluid of the patients with premature rupture of the membrane (PROM). In the present study we examined the effect of IL‐1 on the fetal keratinocytes, one of the main tissue exposed to the increased IL‐1. Exposure of IL‐1 at over 0.1 pg/ml IL‐1 significantly (p<0.01) inhibited proliferation of the keratinocytes in low calcium keratinocyte growth medium obtained from the umbilical cord. Immunohistochemical analysis using antibodies to various cytokeratins revealed that the umbilical cord keratinocytes is more similar to the mucosal epithelium than the skin epithelium. These results suggest that IL‐1 α increased in amniotic fluid in the PROM cases leads the immature proliferating mucosal epithelium to maturate for protection of the fetus against environment.
Stimulation of fibronectin synthesis through the protein kinase c signaling pathway in normal and transformed human lung fibroblasts
Authors: Lee Byung‐Heon , Park Rang‐Woon , Choi Je‐Yong , Ryoo Hyun‐Mo , Sohn Kun‐Young , Kim In‐San
Published: July 2007

We examined the role of the protein kinase C (PKC) signaling pathway in the stimulation of fibronectin synthesis in both normal and transformed human lung fibroblasts. Phorbol myristate acetate (PMA), a potent PKC activator, stimulated fibronectin synthesis in both normal and transformed fibroblasts in a time and dose dependent fashion. Down‐regulation of PKC by prior exposure of cells to a high concentration of PMA blocked the increase in fibronectin synthesis and mRNA levels induced by PMA. Bisindolylmaleimide, a specific inhibitor of PKC, also abolished the PMA‐induced fibronectin synthesis. 4α‐phorbol didecanoate, an inactive phorbol ester, failed to affect fibronectin synthesis. These data suggest that PMA stimulates fibronectin synthesis and gene expression through the PKC signaling pathway in both normal and transformed human lung fibroblasts.
Yeast thiol‐dependent protector protein expression enhances the resistance of Escherichia coli to hydrogen peroxide
Authors: Ahn Soo Mi , Lee Soo Min , Chung Taewan , Kim Kanghwa , Park Jeen‐Woo
Published: July 2007

A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol‐containing oxidation system but not against an oxidation system without thiol. This 25‐kDa protein was thus named thiol‐dependent protector protein (TPP). The role of TPP in the cellular defense against oxidative stress was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPP (strain YP) and mutants in which the catalytically essential amino acid cysteine (Cys‐47) has been replaced with alanine (strain YPC47A) or tryptophan (Trp‐82) has been replaced with phenylalanine (strain YPW82F) by a site‐directed mutagenesis. There was a distinct difference between these three strains in regards to growth inhibition kinetics, viability, modulation of activities of superoxide dismutase and catalase, and the accumulation of oxidized proteins. These results suggest that TPP may play a direct role in the cellular defense against oxidative stress by functioning as an antioxidant protein.
Expression, purification and properties of recombinant E. coli ribonuclease III
Authors: Srivastava Neelam , Srivastava Rai Ajit K.
Published: July 2007

Cloned RNase III gene in a T7 RNA polymerase promoter system was expressed in Escherichia coli cells lacking endogenous RNase III, and the over‐expressed recombinant RNase III was purified to homogeneity using ion exchange, exclusion and affinity column chromatography. The overexpressed RNase III was found to separate with the membrane fraction after sonication, which was solubilized, fractionated with (NH)2SO4 and the active fractions used for further purification. The properties of the purified recombinant RNase III were studied using the synthetic RNA substrate, 3[H]poly[A].poly[U], and the natural substrates, 7S and p10Sa RNAs, and compared with the partially purified RNase III from wild‐type E. coli cells. The recombinant RNase III showed maximal activity at 37°C and at a pH range of 6.9 to 7.4, which was similar to the RNase III purified from the wild‐type cells. Recombinant RNase III efficiently hydrolyzed 3[H].poly[A].poly[U] in the presence of Mg2+. However, the recombinant RNase III cleaved natural RNA substrates efficiently and accurately in the presence of Mn2+. A concentration of Mn2+ ranging from 150 to 300 µM was found to be optimal; concentrations higher than 0.5 mM were inhibitory. Other divalent cations did not support RNase III activity. Monovalent cations, Na+, K+ and NH4+ at 20 mM were equally effective in stimulating RNase III activity although they were not absolutely required for the activity. The thermal stability of the recombinant RNase III was examined at two temperatures, 37° and 50°C. Incubation of RNase III at 37°C for 30 min did not affect activity, but it lost almost 50% of its activity when incubated at 50 °C for 30 min. Thus, the recombinant RNase III prefers Mn2+ for the cleavage of natural substrates and exhibits several properties similar to the wild‐type RNase III.
Cystatin‐like domain of mouse countertrypin, a member of mammalian fetuin family, is responsible for the inhibition of trypsin. Evidence from site‐directed mutagenesis
Authors: Yoshida Koji , Suzuki Yasuyuki , Yamamoto Kazuhiko , Sinohara Hyogo
Published: July 2007

Members of mammalian fetuin family, such as human α2HS glycoprotein and bovine fetuin, consist of three domains, two tandemly arranged cystatin‐like domains and an unrelated domain, but they have no inhibitory activity against cysteine proteinases. We found that countertrypin, a novel trypsin inhibitor, is mouse counterpart of human α2HS glycoprotein, and that human α2HS glycoprotein and bovine fetuin which were prepared without use of ethanol are capable of inhibiting trypsin (Yamamoto, K. and Sinohara, H. (1993) J. Biol. Chem, 268, 17750‐17753). In the present study, cDNA encoding countertrypin was isolated and sequenced, and evidence is presented, based on the site‐directed mutagenesis, that lysine‐231 in the second cystatin domain is the P1 site for trypsin inhibition.
Cytochrome P450scc spin state transitions in the thin solid films
Authors: Guryev O. , Erokhin V. , Usanov S. , Nicolini C.
Published: July 2007

Langmuir‐Blodgett films of cytochrome P450scc were prepared on the solid supports and their spectral properties were investigated. Being immobilized, hemoprotein changes its spin state from initially high to low spin. This transition is reversible since after the solubilization of hemoprotein, the spin state equilibrium is shifted towards high‐spin state. Anaerobic reduction of film incorporated cytochrome P450scc by electron transfer chain (NADPH→adrenodoxin reductase→adrenodoxin) revealed the low rate of the reaction that coincides well with the content of the hemoprotein low‐spin form. We suggest that particularly regular orientation of solid cytochrome P450scc are of crucial importance for this phenomenon.
Stem cell factor protects bone marrow‐derived cultured mast cells (BMCMC) from cytocidal effect of nitric oxide secrected by fibroblasts in murine BMCMC‐fibroblasts coculture
Authors: Park Sung‐Joo , Jun Chang‐Duk , Choi Byung‐Min , Lee Eun‐Jung , Kim Hyung‐Ryong , Cho Hae‐Won , Chung Hun‐Taeg
Published: July 2007

The survival of mast cells are dependent on two kinds of growth factors, one derived from T cells (IL‐3) and another derived from fibroblasts (stem cell factor [SCF]). The 3T3 fibroblast cell line derived from WCB6Fl‐+/+ mouse embryos (+/+ 3T3 fibroblasts) supported the proliferation of bone marrow‐derived cultured mast cells (BMCMC) in the PWM‐stimulated spleen cell conditioned medium (PWM‐SCM), whereas the 3T3 fibroblast cell line from WCB6Fl‐Sl/Sld mouse embryos (Sl/Sld 3T3 fibroblasts) did not. To study the role of nitric oxide (NO) on the growth of mast cells in BMCMC‐fibroblasts coculture, we used a NO synthase inhibitor, NG‐monomethyl‐L‐arginine (NGMMA). NGMMA recovered survival and maintained proliferation of mast cells in BMCMC‐Sl/Sld 3T3 fibroblasts coculture. Sl/Sld 3T3 fibroblasts as well as 3T3 fibroblasts from NIH‐+/+, BALB‐+/+ or Swiss‐+/+ mouse embryos secreted NO in PWM‐SCM, but not in α‐MEM. SCF protected BMCMC from cytotoxicity of exogenous NO in IL‐3‐supplemented α‐MEM. We concluded that SCF might protect BMCMC from cytocidal effect of NO in BMCMC‐fibroblasts coculture.
Erythrocyte membrane Na,K ATP ase activity can be a marker of liver histopathology
Authors: Kuralay Filiz , Tanyalçin Tijen , Kutay Fatma , Yuce Gül , Ersoz Galip , Batur Yücel
Published: July 2007

Erythrocyte membrane Na+,K+; Ca2+ ATP ase activities, cholesterol (CH) phospholipid (PL) composition and erythrocyte glutathione (GSH) contents were determined in controls, in patients with chronic active hepatitis and liver cirrhosis. Na+,K+ ATP ase activities were significantly (P<0.0001) less in patients with chronic active hepatitis and liver cirrhosis (n=8, 0.102 ± 0.02 µmol P/mg protein/hour; n=8, 0.081 ± 0.02 µmol P/mg protein/hour) than in controls (n=10, 0.219 ± 0.05). Histopathological analysis of liver sections obtained from patients with chronic active hepatitis (n=3) and liver cirrhosis (n=2) correlated well with erlthrocyte biochemical findings. There was a significant negative correlation between Na+,K+ ATP ase activity and portal fibrosis (P<0.05, r=‐8680). However, further experiments performed on larger study populations are needed to better elucidate this correlation. Therefore, Na+,K+ ATP ase activity measurement can be reliable assessment of liver fibrosis.
Purification and enzymatic peptide mapping of protein synthesis elongation factor‐2 from mink and chicken livers
Authors: Riis Bent
Published: July 2007

This investigation has shown it is possible to purify elongation factor‐2 from livers of two rather distinct animal species, minks and chicken, to high homogeneity by employing the same purification procedure. It is also shown that making peptide maps of the factor by the use of Staphylococcus aureus Endoprotease Glu‐C gives the same pattern. Combined, these two experimental approaches show that the evolution has conserved this elongation factor in two species with a rather different physiology and choice of feeding substances. It is also shown that using mink or chicken livers as staring material for purification of eEF‐2 is both technically and economically sound when obtaining large amounts of highly purified eEF‐2 is the ultimate goal.
The amino acid sequence of the spinach basic cupredoxin plantacyanin
Authors: Mann Karlheinz , Eckerskorn Christoph , Mehrabian Zara , Nalbandyan Robert M.
Published: July 2007

We describe the amino acid sequence of plantacyanin isolated from spinach leaves. This small basic copper‐binding plant cupredoxin contains 91 amino acids (accession no.S70746) and its theoretical average mass of 9788.2 Da was confirmed by electrospray ionization mass spectrometry. When compared to the sequences of other plant cupredoxins, the most similar homologs were cucumber basic blue protein and stellacyanin from Laquer tree with 44‐58% identical residues. Other known plant cupredoxins were significantly less similar which may allow to divide the plant cupredoxins into two subgroups, the single‐domain cupredoxins (plantacyanin, cucumber basic blue protein and stellacyanin) and the two‐domain, hydroxyproline‐containing cupredoxins (cucumber peeling cupredoxin, basic blue protein from Arabidopsis thaliana and horseradish umecyanin).
Effect of cycloheximide on the expression of LPS‐inducible iNOS, IFN‐β, and IRF‐1 genes in J774 macrophages
Authors: Hattori Yoshiyuki , Akimoto Kazumi , Matsumura Michiko , Tseng Chen‐Chiang , Kasai Kikuo , Shimoda Shin‐Ichi
Published: July 2007

The effect of cycloheximide (CHX) on the gene expression for inducible NO synthase (iNOS), interferon (IFN)‐β, and IFN regulatory factor (IRF)‐1 was examined in LPS‐stimulated J774 macrophages. LPS caused increased expression of mRNAs specific for iNOS, IFN‐β, and IRF‐1 with different kinetics. Addition of CHX resulted in inhibition of the LPS‐induced iNOS gene expression and parallel decrease in NO production. In contrast, expression of IFN‐β and IRF‐1 genes in response to LPS was potentiated in the presence of CHX. These results indicate that de novo protein synthesis is not required for IFN‐β and IRF‐1 gene expression and that ongoing protein synthesis including IFN‐β and IRF‐1 may be involved in the induction process of iNOS in mouse macrophages.
Transblot identification of avidin‐interacting proteins in rat salivary glands
Authors: Shirakawa Satoko , Murata Takatoshi , Mochizuki Hajime , Takehara Tadamichi , Kobayashi Shigeru , Haneji Tatsuji
Published: July 2007

Blotting of rat parotid gland proteins separated by SDS‐PAGE and transferred to Immobilon transfer membranes revealed that avidin‐peroxidase conjugate interacted with bands having estimated molecular weights of 72, 74, and 120 kDa. Even at the lowest concentration of avidin‐peroxidase used in the general ABC method (1:2000 dilution), three bands were clearly discernible. The staining reaction of parotid gland proteins was eliminated on preincubating the proteins with native avidin. The staining reaction was markedly reduced with the proteins obtained from submandibular/sublingual glands.
The role of multivalent cations in oligonucleotide cellular uptake
Authors: Wu‐Pong Susanna
Published: July 2007

Oligodeoxynucleotides (ONs) are a powerful new class of drugs whose transport to the cytoplasm or nucleus inefficient and incompletely defined. Thus, to further extend our understanding of the mechanism of ON cellular uptake, the role of multivalent cations in ON cellular uptake was examined. All the multivalent cations tested, except magnesium, significantly increase ON uptake and was predictably related to the cation's electronegativity but only partially due to an increase in surface‐binding. In addition, the primary mechanism of uptake is largely independent of trypsin‐sensitive surface components, cellular energy, and several specific, calcium‐dependent cellular processes. Therefore, On cellular uptake may involve an interaction between multivalent cations and either the ON, the cell membrane, or both.
Mitochondrial NADH dehydrogenase activity and ability to tolerate acetaldehyde determine faster ethanol production in Saccharomyces cerevisiae
Authors: Shankar C. S. , Ahamad P.Y. Aneez , Ramakrishnan M. S. , Umesh‐Kumar S.
Published: July 2007

In rapidly fermenting yeast, the rotenone insensitive mitochondrial NADH dehydrogenase was not completely repressed by high glucose. This activity appeared to enhance the glycolytic rate due to which acetaldehyde accumulated intracellularly. To overcome the toxicity of acetaldehyde, the strain produced stress proteins. During late stationary phase of growth, the accumulated acetaldehyde was converted to ethanol resulting in faster ethanol production.
Cellular and subcellular localization of glycogenin in chicken retina
Authors: Miozzo Maria C. , Maldonado Cristina , Curtino Juan A.
Published: July 2007

We investigated the cellular and subcellular localization of glycogenin in chicken retina using a polyclonal antibody raised against chicken muscle glycogenin. The antiserum recognized both free and glycogen‐bound glycogenin on dot blots. Immunocytochemistry revealed an uniform staining of all the retina layers except for the outer segments and ganglion cells layers that were very weakly stained and for the inner segments layer and a zone between the inner nuclear and inner plexiform layers that were heavily stained. Electron microscopy of neuronal cells showed immunoreactivity localized in the cytoplasm and nucleus. This is the first description of the cellular and subcellular localization of glycogenin. Our results suggest that the biosynthesis of glycogen could begin in both, the cytoplasm and nucleus of the neurone.
Characterization of the C‐terminal domain of ras‐GTPase‐activating protein (ras‐GAP) as substrate for epidermal growth factor receptor and p60c‐src kinase
Authors: Borowski Peter , Kornetzky Lutz , Heiland Max , Roloff Sandra , Weber Wolfgang , Laufs Rainer
Published: July 2007

We describe in vitro tyrosine phosphorylation of the C‐terminal 334 amino acids of ras‐GTPase‐activating protein (ras‐GAP)1 that contains the activity domain for ras interaction. To date, there have been no other phosphorylation sites determined than the reported in N‐terminal domain of ras‐GAP Tyr‐460, which is considered to be the major phosphorylation site of ras‐GAP. In our assays some differences of the kinetic parameters were observed when the reaction was catalyzed by EGF‐R compared to p60c‐src. Enzyme specific regulation of activity is associated with autophosphorylation which leads to reduced (in case of EGF‐R) or increased (in case of p60c‐src) phosphorylation of the C‐terminal 334 amino acids of ras‐GAP (GAP334). Because of the characteristics of these investigated reactions the phosphorylation of GAP334 seems to be ‐ independent from the presence of SH2 or SH3 domains ‐ triggered off by complex mechanisms different from those regulating the phosphorylation at Tyr‐460.
Effects of 2‐deoxy‐D‐glucose on the functional state of the rat myoblast GLUT 1 transporter
Authors: Mesmer Ogubay T. , Lu Zhuomei , Xia Ling , Lo Theodore C. Y.
Published: July 2007

Based on the rationale that internalized 2‐deoxy‐D‐glucose (dGlc) is toxic to cells, glucose transport (GLUT) defective myoblast mutants have been isolated by their ability to grow in glucose‐free medium containing dGlc. Recent studies revealed that the GLUT 1 transport process was activated when GLUT 3‐ GLUT 4‐ mutants were grown in glucose‐free medium [1]. It was therefore puzzling why these GLUT3‐ GLUT4‐ myoblasts could survive in the presence of dGlc during the mutant selection process. The present study revealed that GLUT 1 transport affinity in dGlc‐grown cells was at least four folds lower than that in control cells. This loss of GLUT 1 transport activity was apparent only after exposure to the toxic sugar analogues for more than 10 hrs. This dGlc‐mediated effect was not due to competitive inhibition by the residual dGlc carried over from growth medium, changes in glycolytic enzymes, nor accumulation of the negatively charged dGlc‐6‐PO4. In fact, GLUT 1 transcript level was elevated in dGlc‐treated cells. Both immunoprecipitation and immunoblotting studies indicated that the size of the GLUT 1 transporter in dGlc‐grown myoblasts was reduced from 52 kDa to that of the unglycosylated form (38 kDa). These findings suggest that growth in the presence of dGlc inhibits glycosylation of the GLUT 1 transporter, thus reducing its transport affinity. This inability of the GLUT 1 transporter to take up dGlc may therefore explain why GLUT 3‐ GLUT 4‐ mutants are able to grow in the presence of the toxic dGlc during the mutant selection procedure.
Structure of the N‐terminus of the Escherichia coli ribosomal protein L7/L12
Authors: Todorova Roumiana T.
Published: July 2007

Three mutant forms of the ribosomal protein L7/ L12 (S1Y, M14Y and M26Y) were constructed in order to use the Tyr residues as internal labels at the N‐terminal region. The proteins were studied by the methods of 1H‐NMR, Fluorescence, Circular dichroism spectroscopy and Differencial scanning calorimetry. The secondary structure of N‐terminus (residues 1‐36) was predicted by the programme “Globule” [1, 2]. Y1 is located in a structurally disordered region at the free end of the molecule. M14 and M26 are located in structurally ordered regions. The replacement M14 does not change the structure of the protein. The replacement M26 disturbs the helicity in the region of the mutation. The first 1‐5 positions from the N‐terminus of L7/ L12 are disordered, the positions 7‐14 participate in an α‐helical/ β‐structural region and positions 19‐30 in a strongly α‐helical region.
Dominant negative effect of the truncated p110 subunit of phosphatidylinositol‐3 kinase
Authors: Takayanagi Junji , Kimura Koutarou , Nishioka Naoya , Akimoto Kazunori , Moriya Shigeharu , Ohno Shigeo , Fukui Yasuhisa
Published: July 2007

Effects of p110, the catalytic subunit of PI‐3 kinase, on induction of TPA response element‐driven promoter by EGF was examined. The induction was enhanced by co‐expression of the wild type p110. The truncated p110 mutants containing the binding site for p85 but missing the catalytic activity repressed the induction. A mutant with no binding activity to p85 did not show this effect. These results suggest that PI‐3 kinase is involved in signal transduction of EGF and that the truncated p110s capable of binding to p85 serves as a dominant negative reagent for PI‐3 kinase.
The methylation pattern of a cytosine DNA‐methyltransferase gene in Arabidopsis thaliana plants
Authors: Kutueva Lyudmila I. , Ashapkin Vasili V. , Vanyushin Boris F.
Published: July 2007

Using a PCR‐amplified 5′‐end proximal 600‐bp fragment and two cDNA clones of cytosine DNA‐methyltransferase gene of Arabidopsis thaliana as specific probes in hybridization with plant DNA samples, hydrolyzed by methylation‐sensitive restriction endonucleases HpaII and MspI, it has been established that CCGG sites located in the 5′‐end proximal part of cytosine DNA‐methyltransferase gene are highly methylated at internal C and less but detectably methylated at external C residues. On the contrary, all CCGG sites in 3′‐terminal half of the coding region were found to be unmethylated at both external and internal C residues. No significant differences between methylation patterns of cytosine DNA‐methyltransferase gene in various organs (leaf, stem, flower) of the Arabidopsis thaliana plant were detected.
Effect of thiol reagents on functional properties and heme oxidation in the hemoglobin of Geochelone carbonaria
Authors: Torsoni M. A. , Viana R. I. , Barros B. F. , Stoppa G. , Cesquini M. , Ogo S. H.
Published: July 2007

The reaction of thiol reagents with G. carbonaria hemoglobin was studied, and the oxygen equilibrium and kinetic of oxidation of derivatives determined. The oxygen affinity and kinetic of oxidation of hemoglobin derivatives were modified to various extents depending on the nature of thiol reagents used. Diamide yielded approximately 80% polymeric hemoglobin, although the oxidation kinetic, and the functional properties, were practically invariant (T1/2=10.0 min.; P50 = 5.0 mm Hg at pH 7.4; alkaline Bohr effect = ‐0.64). Iodoacetamide did not modify the electrophoretic pattern significantly, although all the free SH groups of hemoglobin were alkylated. A P50 of 2.5 mmHg at pH 7.4 and the Bohr effect of ‐0.15 were obtained; the T1/2 of about 6.4 min. was shorter than that for un‐modified Hb. Similar T1/2 were obtained for Hb treated with oxidized glutathione, which produced polymeric Hb and glutathionyl‐Hb. The oxygen binding characteristics showed that both of Hb derivatives, glutathionyl‐Hb and polymeric Hb, maintain the capacity to transport the gas.
Description of a one step staggered reannealing method for directional cloning of PCR‐generated dna using sticky‐end ligation without employing restriction enzymes
Authors: Ailenberg Menachem , Silverman Mel
Published: July 2007

A novel method is described for ligation of PCR products into vectors. It utilizes formation of sticky end sequences compatible with those generated on the plasmid. Four primers are used: two primers are designed to contain the desired sequence of the sticky end plus the sequence of the gene and the other two primers contain the same sequence as the first two primers without the additional bases. The primers are paired to create two PCR products containing the additional bases in a staggered 5′ position. Following melting and reannealing, the newly formed products contain the additional sequences as sticky overhangs compatible with the sticky ends of the plasmid.
Disulfide bond formation between the N‐terminal region of p56lck and the cytoplasmic domain of CD8 studied by Electrospray Ionization and Matrix‐Assisted Desorption/Ionization time‐of‐flight mass spectrometry
Authors: Yi Gwan‐Su , Cheong Chaejoon
Published: July 2007

Masses of the complexes formed between the C‐terminal region of CD8α (CD8αC) and the N‐terminal region of p56lck (p56lckN) were measured by using Electrospray Ionization (ESI) and Matrix‐Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry (MS). The two peptides were incubated with various metal ions, Zn++, Cd++, Co++, Fe++ and Ca++. The complex formed from the incubation did not contain any metal ions. Instead, its mass suggested that the complex was formed by two disulfide bonds. The fact that the incubation of the complex with Dithiothreitol broke the complex confirmed into monomers that the complex was formed through disulfide bonds. The only disulfide‐mediated complex formed was hetero‐dimer (CD8αC‐p56lckN) and none of homo‐dimers (CD8αC‐CD8αC or p56lckN‐p56lckN) were observed from ESI and MALDI‐TOF mass spectrometry.
Opposite effects of basic fibroblast growth factor and heparin on heparin/heparan sulfate degrading enzymes from Flavobacterium heparinum
Authors: Cavari Simone , Vannucchi Simonetta
Published: July 2007

Heparinase I is normally inhibited by an excess or substrate; in this paper we describe that bFGF counteracts the inhibiting effect of the substrate and that heparinase I activity is increased in the presence of bFGF. This effect was observed using heparin concentrations ranging from 10‐9 to 10‐7 M with either a ten fold molar excess or an equimolar concentration of bFGF. In addition, the degree of depolymerization of heparan sulfate produced by heparitinase from Flavobacterium heparinum was increased in the presence of bFGF.
Elastin‐derived peptide induces monocyte chemotaxis by increasing intracellular cyclic GMP level and activating cyclic GMP dependent protein kinase
Authors: Uemura Yuko , Okamoto Kouji
Published: July 2007

An elastin‐derived peptide with an average molecular mass of 25 kDa was shown to induce monocyte chemotaxis at the optimal concentration of 10‐1 μg/ml. Homologous deactivation test showed that monocytes exposed to the elastin‐derived peptide at 10‐1 μg/ml lost their chemotactic responsiveness when reexposed to the same stimulus. In conjunction with chemotactic response to the elastin‐derived peptide, intracellular guanosine 3′, 5′‐monophosphate (cGMP) levels were enhanced but intracellular adenosine 3′, 5′‐monophosphate (cAMP) levels were not. The monocyte migration induced by the elastin‐derived peptide was inhibited by cGMP dependent protein kinase (PKG) inhibitor, but not by cAMP dependent protein kinase inhibitor and protein kinase C inhibitor. These results suggest that the elastin‐derived peptide induces monocyte chemotaxis by increasing the level of cGMP, followed by activating PKG.
The growth kinetics of intracellular bacille calmette‐ guerin (BCG) within human monocyte‐derived macrophages of different ethnic populations
Authors: Fazal Nadeem
Published: July 2007

In this paper we have examined intracellular mycobacterial growth in human monocyte‐derived macrophage cell cultures. Cells from three population groups were selected to compare and contrast the kinetics of intracellular BCG growth both by determining changes in metabolic activity of dividing bacteria by radioisotope incorporation and growth/survival by estimating colony forming unit by Fazal's Microcolony Counting Method. These data indicate that BCG infects and multiplies intraceltularly in human macrophages and may serve as an in vitro model for studying inherent capability of mycobacteria to grow and infect macrophage populations. There is however, no inhibition or reduction of intracellular mycobacterial growth from a Caucasian male and female, and an Afrocarribean female donor macrophages. On the contrary, there is a ten‐fold increase in the replication of mycobacteria over a 7‐day infection period. Patterns of BCG growth within macrophage cultures from different donors were determined and variation calculated at different days post‐infection. Experiment to experiment coefficient of variation of intracellular BCG growth estimates for a single donor is between 4‐12%. In contrast, the coefficient of variation of intracellular BCG growth between three different macrophage donors studied is <4%.
Ursodeoxycholate promotes protein phosphorylation in the cytosol of rat hepatocytes
Authors: Capuano Ferdinando , Barone Michele , D'Eri Nicola , Russo Elisabetta , Varone Davide , Francavilla Antonio , Papa Sergio
Published: July 2007

A study is presented of the effect of the bile salt ursodeoxycholate (UDC) on protein phosphorylation by [γ‐32P]ATP in the cytosol from ra hepatocytes. Gel electrophoresis and corresponding autoradiograms of cytosolic proteins show that UDC promotes phosphorylation of at least eight different protein bands. Four of them (the 36, 60, 64 and 76 kDa) are phosphorylated by Ca2+ and phospholipid‐dependent protein kinase (PKC); three (the 31, 51 and 71 kDa) are phosphorylated by cAMP‐dependent protein kinase (PKA) and one protein band, with molecular weight of 34 kDa, apparently contains substrates of both PKC and PKA. Data are reported indicating that UDC can directly affect the intrinsic activity of protein kinases.
The Presence of A manganese‐rich particle in lysosome of rat pancreas due to excess manganese treatment
Authors: Okamoto Yoshihiro , Oshima Reiko , Inagaki Kazuhiro , Aita Saburo , Nisioka Hideo , Kondo Yumi , Ishizuka Hiroshi , Takada Jitsuya , Nishida Mikio
Published: July 2007

Manganese (Mn) accumulates at a higher level in the pancreas than in any other organs when excess Mn is administered to the rat. The present study is carded out to analyze the intracellular localization of Mn existed in the pancreatic cell of Mn‐treated rats. Transmission electron microscope and X‐ray micro‐analysis connected with a rapid freezing fixation technique showed that a large amount of Mn was localized in tysosomal particles of the pancreatic cell of Mn‐treated rats. The Mn‐rich particles disappeared when the element‐administration was discontinued, showing that the accumulation of Mn is reversible. To confirm that Mn is in the lysosomes, a centrifugal subcellular‐fractionation and a neutron activation analysis were carried out. The result indicated that much Mn existed in the lysosomal fraction.
Characterization of phosphate transport in Strephtomyces granaticor
Authors: Lichá I. , Beneš I. , Janda S. , Hošt′álek Z. , Janáček K.
Published: July 2007

Transport of inorganic phosphate in Streptomyces granaticolor was characterized in two growth stages; kinetic parameters were determined and two transport systems were found in both stages, with the following values: KT1=0.06 mM, Jlim1=0.95 nmo1 min‐1 (mg DS) ‐l, and KT2=1.80 mM, Jlim2= 25 nmol min‐1 (mg DS)‐1. Both systems require metabolic energy and substrates, such as sugars or polyols; when alanine was used or the energy source was omitted, the kinetic parameters changed in both systems. Both systems were inhibited by the ionophore CCCP with identical Ki. KCN, an inhibitor of terminal cytochrome oxidase, inhibited the uptake of phosphate only partially, the uptake was inhibited completely when also the inhibitor of the alternative oxidative pathway (salicylhydroxamic acid) was added. Antimycin A inhibited the uptake completely. Arsenate inhibited competitively.
Properties of Thermus aquaticus β‐NADH oxidase immobilised on various supports
Authors: Sanjust Enrico , Curreli Nicoletta , Rescigno Antonio , Bannister Joe V. , Cocco Dina
Published: July 2007

β‐NADH oxidase purified from Thermus aquaticus was covalently immobilised on various solid supports. The preparations obtained were compared with the soluble enzyme for activity and kinetic properties. Activated glutaryl‐PVA was found to be the best support. The immobilised enzyme was less stable at high temperatures than the soluble enzyme. No differences could be detected in the presence of organic solvents.
Molecular cloning of the leub genes from Mycobacterium bovis BCG and Mycobacterium tuberculosis
Authors: Han Mi Young , Son Mi Young , Lee Seung‐Hwan , Kim Jin Koo , Huh Jae Suk , Kim Jeong Hyun , Choe In Seong , Chung Tai Wha , Choe Yong‐Kyung
Published: July 2007

A gene responsible for the biosynthesis of leucine has been cloned by the complementation of the Escherichia coli leuB6 auxotroph mutant after transformation with the Mycobacterium bovis BCG genomic DNA library, which was constructed by ligating the partially digested BCG DNA with Sau3AI into the pUC 19 digested with BamHI. Sequencing of the leuB gene of BCG revealed an ORF (open reading frame) of 1,011 bp encoding isopropytmalate dehydrogenase with a calculated molecular weight of 42 kDa. The leuB gene of Mycobacterium tuberculosis isolated from Korean tuberculosis patient is shown to be identical to that of BCG except one bp.
Intracellular calcium variation in heparin‐capacitated bovine sperm
Authors: Córdoba Mariana , Santa‐Coloma Tomás A. , Beorlegui Norma B. , Beconi Martha T.
Published: July 2007

This report investigates the mechanisms by which heparin induces capacitation of sperm. Capacitation was determined with chlortetracycline and intracellular calcium concentration, [Ca2+]i, with FURA 2‐AM. Atter 15 minutes incubation with heparin, [Ca2+]i was increased 60% over basal, reaching a plateau thereafter. Sixty percent of calcium entry was inhibited by methoxy‐verapamil, suggesting that activation of voltage dependent calcium channels (VDCC) may be involved in the process. A significant correlation (R2 = 0.88, p<0.006) was found between the percentage of capacitated spermatozoa and the [Ca2+]i increase. The effects of heparin on both processes were blocked by the protein kinase C (PKC) inhibitors staurosporine (100%) and GF‐109203X (90%). It is concluded that heparin may induce sperm capacitation and calcium influx mainly through VDCC (approx. 60%), and also through other membrane systems (40%). Both systems of calcium entry as well as the capacitation process appear to involve on PKC activity.
Carbohydrate handling in the hind leg muscle of exercising rats
Authors: Adán C. , Ardévol A. , Remesar X. , Alemany M. , Fernández‐López J. ‐A.
Published: July 2007

The arterio‐venous concentrations of oxygen, glucose and lactate, as well as blood flow and muscle levels of lactate, glucose, hexose‐phosphates and glycogen of rat hind leg muscle were determined under basal conditions and under mild and intense exercise, as well as during post‐exercice recovery. During intense exercise and fatigue glycogen is practically exhausted, providing glycosyl residues to the hexose‐phosphates pool in addition to increased glucose uptake from the blood. The result is the production of huge amounts of lactate, which accumulates in muscle and the skin, and buildup the arterial concentrations. During recovery lactate is slowly disposed of, and the muscle takes up large amounts of glucose which is stored into glycogen, with fully reinstated glucose oxidation. The data shown suggest that the shift from oxidative to mainly anaerobic utilization of glucose is not as streamlined as is usually assumed, since the results found here hint at the wasteful utilization of glycogen‐derived hexose skeletons for other synthetic pathways. Glucose, nevertheless, is of paramount importance as energy staple to sustain untrained intense exercise in the rat
Molecular cloning and analysis of a HSP (heat shock protein) ‐ Like 42 kDa antigen gene of Mycoplasma hyopneumoniae
Authors: Chou Shih‐Yu , Chung Tung‐Liang , Chen Re‐Jen , Ro Lee‐Hwa , Tsui Pei‐I , Shiuan David
Published: July 2007

The recombinant clone expressing the 42 kDa protein (P42) of Mycoplasma hyopneumoniae in Escherichia coli was analyzed. The 4.4 kb HindIII‐XmaI DNA fragment expressing the p42 gene product encodes three ORFs: p42 and pl6 in the forwarding strand, p24 in the reverse strand. Sequence comparisons revealed that p42 could be part of a p65 gene, and has 62% identities with Mycoplasma genitalium HSP70 gene and 56% identities with Bacillus subtilis dnaK gene; p16 and p24 genes share 73% and 47% identities with Erysipelothrix rhusiopathiae dnaJ gene and Pseudomonas fluorescens uvrC gene, respectively. Further analysis demonstrated that P42 is indeed a heat shock protein and the monospecific antibodies against P42 can block the growth of Mycoplasma hyopneumoniae.
Protease triggers dephosphorylation of cdc2 kinase during starfish oocyte maturation
Authors: Sawada Hitoshi , Yokoyama Kuniko , Morinaga Chikako , Yokosawa Hideyoshi , Sawada Michiko Takagi
Published: July 2007

We previously presented evidence that a Z‐Phe‐Ser‐argininal‐susceptible protease which is involved in oocyte maturation of the starfish, Asterina pectinifera is the proteasome (Takagi Sawada et al., Dev. Biol. 150, 414‐418 (1992)). In the present study, we investigated the timing of the function of and the role of the protease in oocyte maturation using Z‐Phe‐Ser‐argininal. By adding the inhibitor in maturing oocytes at various times after 1‐methyladenine treatment, the inhibitory ability was markedly reduced in half the time required for germinal vesicle breakdown. Furthermore, the inhibitor potently blocked the activation of histone H 1 kinase and the dephosphorylation of cdc2 kinase during oocyte maturation. These results indicate that the Z‐Phe‐Ser‐argininal‐susceptible protease, probably the proteasome, plays a key role in the step of the signal transduction pathway that triggers the dephosphorylation of cdc2 kinase in response to the maturation‐inducing hormone.
Effect of long time storage on cytosolic glutathione S‐transferases
Authors: Peters Wilbert H. M. , Roelofs Hennie M. J.
Published: July 2007

Glutathione S‐transferases are a family of enzymes catalyzing the conjugation of glutathione to a wide variety of compounds. Stability of human liver cytosolic glutathione S‐transferase enzyme activity and levels of glutathione S‐transferase class Alpha and Mu were assessed. Cytosolic fractions of sixteen different patients were stored up to two years at three different temperatures: ‐20°, ‐80° and ‐196° Celsius. Samples were assayed for glutathione S‐transferase enzyme activity and glutathione S‐transferase class Alpha and Mu levels, after 3, 6, 12 and 24 months of storage.

No significant differences in glutathione S‐transferase enzyme activity as well as in isoenzyme levels were found during this 24 months of storage at the three different temperatures. It is concluded that stability of glutathione S‐transferases allows long term storage even at ‐20°C, without significant loss of enzyme activity or enzyme protein.
Effect of cyclins and Cdks on the cyclin B1 promoter activation
Authors: Moro A. , Zerfass K. , Joswig S. , Jansen‐Duerr P.
Published: July 2007

To study the effect of several cyclins on cyclin B1 promoter activation, we co‐transfected cyclin A, cyclin E and cyclin D1 expressing plasmids with a cyclin B1 promoter construction driving a luciferase repotnter gene into NIH 3T3 cells. All three cyclins produced activation of the reporter gene, however, co‐transfection of cyclin A with a Cdk2 dominant negative mutant blocks activation by cyclin A. Our results suggest that cyclin B1 activation depends on Cdk2 kinase activity associated to cyclin A. To our knowledge this is the first report showing positive effect of cyclin A, cyclin E and cyclin D1 on cyclin B1 activation and blocking of this activation by a Cdk2 dominant negative mutant.
Purification and characterisation of the soluble glutathione S‐transferase isoenzymes in rat kidney derived NRK cells
Authors: Dierickx Paul J. , Noble Elisabeth
Published: July 2007

Glutathione S‐transferase (GST) enzymes are toxicologically important from many points of view. Rat kidney derived established NRK cells were mass cultured for the isolation of GST isoenzymes. These were enriched by affinity chromatography and separated by chromatofocusing and HPLC. Exactly the same major GST subunits were found in NRK cells as in the rat kidney. Strong evidence was also found for the presence of an aberrant form of GST 7‐7, as was described in rat kidney. A very good correlation between the NRK GST and rat kidney, and especially cis‐platinum treated kidney, was found. It is concluded that NRK cells can be considered as a valuable alternative tool for in vitro research of rat kidney phenomena, especially when toxicological interactions are investigated.
Refolding kinetics of partially reduced and S‐carboxymethylated trypsin‐subtilisin inhibitor from marine turtle eggwhite
Authors: Chaudhuri Tapan K.
Published: July 2007

Three accessible disulphide bonds of basic trypsin‐subtilisin inhibitor from marine turtle eggwhite have been reduced with 0.1M NaBH4 at 0°C under nitrogen atmosphere at pH9.8 and then S‐carboxymethylated. The partially reduced inhibitor retains 80% of the native inhibitory activity towards trypsin and subtilisin. The S‐carboxymethylated inhibitor undergoes slower refolding than the native inhibitor from its fully denatured and reduced state at pH 8.5 in the presence of oxidised and reduced glutathione. The refolding process was characterised by the attainment of the inhibitory activity towards trypsin and subtilisin. The values of the second order rate constant for the refolding reactions of the modified protein are 0.02×102M‐1sec1 and 0.033×102M‐1sec‐1 for its trpsin and subtilisin inhibiting domains and their energies of activation are 20.1 Kcal/mole and 24.6 Kcal/mole. The partially modified inhibitor does not regain complete inhibitory activity even after long incubation in the oxido‐shuffiing buffer. From the above findings it can be concluded that the three disulphide bonds of the native inhibitor are not essential for the inhibitory activity of the trypsinsubtilisin inhibitor but they help in the correct refolding of the inhibitor by forming transient disulphide bonds with the external disulphide reagents as well as with the internal sulphydryl groups.
Complete sequence of the upp gene encoding uracil phosphoribosyltransferase from Mycobacterium bovis BCG
Authors: Kim Jin Koo , Kim Jeong Hyun , Lee Seung‐Hwan , Kang Young , Lim Jong‐Seok , Lee Hee Gu , Kim Sang Jae , Bai Gill Han , Park Young Kil , Cho Sang‐Hyun , Kang Sang Won , Kim Yu Sam , Choe In Seong , Chung Tai Wha , Park Sue‐Nie , Kim Seung Ho , Ahn Jong Seog , Choe Yong‐Kyung
Published: July 2007

The nucleotide sequence of a 799 bp fragment of Mycobacterium bovis BCG containing the putative upp genc that encodes a uracil phosphoribosyltransferase (UPRTase [EC 2.4.2.9]) has been determined. The upp gene of BCG has an open reading frame (ORF) of 621 bp (207 amino acids) starting with GTG (position 112) and ending with TGA (position 733), and its molecular mass was calculated to be 21,864 Da. Comparative analyses of the deduced amino acid sequence of BCG UPRTasc with the UPRTase of six bacterial genera revealed that 24% (50/211) of the residues are perfectly conserved and 32% (67/211) of the residues arc well conserved.
Differences in aldehyde oxidase activity in cytosolic preparations of human and monkey liver
Authors: Sugihara Kazumi , Kitamura Shigeyuki , Tatsumi Kiyoshi , Asahara Toshimasa , Dohi Kiyohiko
Published: July 2007

This study presents data showing individual differences in aldehyde oxidase activity in human and monkey liver cytosols. When assayed with benzaldehyde as a substrate, a significant inter‐subject variation in the activity was found in the human liver preparations. When assayed with N1‐methylnicotinamide as a substrate, the inter‐subject variation of the activity was also observed, but to a lesser extent compared with that of the activity with benzaldehyde. Similarly, variations in aldehyde oxidase activity were found in the monkey liver preparations when assayed with benzaldehyde or N1‐methylnicotinamide. The present study suggested that at least two isozymes of aldehyde oxidase exist in the human liver preparations.
Denaturation of uridine phosphorylase from Escherichia coli K‐12 by guanidine hydrochloride
Authors: Kurganov B. I. , Burlakova A. A. , Chernyak V.Ya. , Debabov V. G.
Published: July 2007

Denaturation of uridine phosphorylase from Escherichia coli K‐12 by guanidine hydrochloride results in red shift of the maximum in the protein fluorescence spectrum, dissociation of the hexameric enzyme molecule into monomers, and the loss of the enzymatic activity. The initial rate of the enzyme reactivation after the dilution of the enzyme preincubated with guanidine hydrochloride has the second order with respect to protein. It is assumed that the rate of the reactivation process is limited by the reassociation of monomers possessing low enzymatic activity to dimers followed by the rapid step of hexamer formation.