bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a novel nanoflow liquid chromatography-parallel reaction monitoring mass spectrometry-based method for quantification of angiotensin peptides in HUVEC cultures
Authors: He Chuan , Hu Simiao , Zhou Wanxing
Published: September 2020

Background

This study aimed to develop an analytical method using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the determination of angiotensin (Ang) I, Ang (1-9), Ang II, Ang (1-7), Ang (1-5), Ang III, Ang IV in human umbilical vein endothelial cell (HUVEC) culture supernatant.

Methods

HUVEC culture supernatant was added with gradient concentrations (0.05–1,000 ng/ml) of standard solutions of the Ang peptides. These samples underwent C18 solid-phase extraction and separation using a preconcentration nano-liquid chromatography mass spectrometry system. The target peptides were detected by a Q Exactive quadrupole orbitrap high-resolution mass spectrometer in the parallel reaction monitoring mode. Ang converting enzyme (ACE) in HUVECs was silenced to examine Ang I metabolism.

Results

The limit of detection was 0.1 pg for Ang II and Ang III, and 0.5 pg for Ang (1-9), Ang (1-7), and Ang (1-5). The linear detection range was 0.1–2,000 pg (0.05–1,000 ng/ml) for Ang II and Ang III, and 0.5–2,000 pg (0.25–1,000 ng/ml) for Ang (1-9) and Ang (1-5). Intra-day and inter-day precisions (relative standard deviation) were <10%. Ang II, Ang III, Ang IV, and Ang (1-5) were positively correlated with ACE expression by HUVECs, while Ang I, Ang (1-7), and Ang (1-9) were negatively correlated.

Conclusion

The nanoflow liquid chromatography-parallel reaction monitoring mass spectrometry-based methodology established in this study can evaluate the Ang peptides simultaneously in HUVEC culture supernatant.
Numerical investigation of microbial quorum sensing under various flow conditions
Authors: Jung Heewon , Meile Christof D.
Published: September 2020

Microorganisms efficiently coordinate phenotype expressions through a decision-making process known as quorum sensing (QS). We investigated QS amongst distinct, spatially distributed microbial aggregates under various flow conditions using a process-driven numerical model. Model simulations assess the conditions suitable for QS induction and quantify the importance of advective transport of signaling molecules. In addition, advection dilutes signaling molecules so that faster flow conditions require higher microbial densities, faster signal production rates, or higher sensitivities to signaling molecules to induce QS. However, autoinduction of signal production can substantially increase the transport distance of signaling molecules in both upstream and downstream directions. We present empirical approximations to the solutions of the advection–diffusion–reaction equation that describe the concentration profiles of signaling molecules for a wide range of flow and reaction rates. These empirical relationships, which predict the distribution of dissolved solutes along pore channels, allow to quantitatively estimate the effective communication distances amongst multiple microbial aggregates without further numerical simulations.
Addition of Mackerel Fish Head Flavor Powder to Cilok's Preference Level
Authors: Azizah Suci Utami Nur , Junianto . , Rostini Iis , Pratama Rusky Intan
Published: September 2020

This research aims to determine the appropriate level of the addition of mackerel fish head flavor powder to obtain the cilok most preferred by panelists. This research was conducted from September to December 2019 at the Fishery Product Processing Laboratory, Faculty of Fisheries and Marine Sciences Universitas Padjadjaran. The method used is experimental with 4 treatment levels of adding mackerel fish head flavor powder that is 0%, 5%, 6%, and 7% of the amount of tapioca flour and wheat flour used. The parameters observed were the level of preference for appearance, aroma, texture, and taste of cilok. Based on the research results it can be concluded that the most preferred cilok was obtained from the treatment of adding mackerel fish head flavor powder as much as 6%.
Analysis of All-Cause Mortality Population of Insurance Applicants
Authors: Ali Muhammad Ghafoor
Published: September 2020

Background: The risk of all-cause mortality is assessed in the study from factors consisting of BMI, medical history, socioeconomic status etc where factors are available in the life insurance organization data and submitted at the time of registration and subsequently in the death claim papers which gives detail about the state of health and cause of death.

Methods: A retrograde study has been carried out of 300 deceased life insured (2005-2014) from the data of a life insurance organization in Pakistan about all-cause mortality from the available factors in death claim files. The research instruments were consisting of death claim register, proposal forms, medical reports and death claim papers.

Results: The results indicate maximum deaths found due to Cardiovascular causes (43.34%) followed by Cancer (9.00%) while minimum found in, Endocrine disorders(0.33%)followed by Respiration(0.67%). However significant causes of death occurred due to Accident, Liver, CNS, Multisystem involvement, Natural disaster and Un- natural. The correlation co-efficient found as ‘0.07’ for all-cause mortality indicating positive relationship between BMI and Life Expectancy.

Conclusion: Medical history plays an important but not a compulsory role in mortality. Since BMI (WHO) showed higher mortality with normal BMI whereas BMI (Asia) showed it at-risk value therefore WHO should make separate criteria of standard of BMI as being different for different ethnicities. In Insurance organization NT-proBNP and BMI of ethnic area be used to asses life expectancy in applicants. Necessary measures should be opted for implementation of “The Sendai Framework for Disaster Risk Reduction 2015-2030”.
Electrogenic proton translocation by the chloroplast cytochrome b6/f complex reconstituted into phospholipid vesicles
Authors: Hurt Eduard C. , Hauska Günter , Shahak Yosepha
Published: July 2002

When the cytochrome b
6/f complex from chloroplasts is incorporated into liposomes, reduction of external plastocyanin by plastoquinol is stimulated under uncoupling conditions. An extra H+/e− is ejected from the vesicles during the reaction, in addition to the scalar proton liberated from plastoquinol. This is stimulated by valinomycin/K+ and abolished under uncoupling conditions. Furthermore, the formation of a membrane potential during the reaction, negative inside the vesicles, is observed with the help of carbocyanine dyes. We conclude that the cytochrome b
6/f complex, like the cytochrome bc
1 from mitochondria, functions as an electrogenic proton translocator.
Cell fusion by spark discharge and its relevance for evolutionary processes
Authors: Küppers G. , Zimmermann U.
Published: July 2002

Electrofusion of mesophyll protoplasts of Avena sativa was induced by using electromagnetic coupling of energy including that from a spark discharge. The results suggest that electrofusion may have played an important role in the evolutionary process and that new species arose in this way by electromagnetic waves emitted by lightning in thunderstorms. If cell fusion had occurred in prehistory by the means described here, then the normally accepted gradual process of evolution could be leap‐frogged. This would explain the eixstence of steps in the fossil record.
Induction of maturation of rat follicle‐enclosed oocyte by forskolin
Authors: Dekel N. , Sherizly I.
Published: July 2002

The diterpene forskolin, which was found to be a potent and reversible activator of adenylate cyclase in intact tissues as well as in broken cell preparations, was employed to investigate the role of cAMP in the induction of oocyte maturation. We have found that forskolin can mimic the effect of LH on the ovarian follicle stimulating both cAMP accumulation and ooctye maturation. These findings suggest that LH‐induced maturation in follicle‐enclosed oocytes is a cAMP‐mediated response.
Reduction of cytochrome b‐561 through the antimycin‐sensitive site of the ubiquinol‐cytochrome c2 oxidoreductase complex of Rhodopseudomonas sphaeroides
Authors: Glaser Elzbieta G. , Meinhardt Steven W. , Crofts Antony R.
Published: July 2002

Cytochrome b‐561 of the ubiquinol‐cytochrome c
2 oxidoreductase complex of Rhodopseudomonas sphaeroides is reduced after flash illumination in the presence of myxothiazol in an antimycin‐sensitive reaction. Flash‐induced reduction was observed over the redox range in which cytochrome b‐561 and the Q‐pool are both oxidized before the flash. The extent of reduction increased with increasing pH, and was maximal at pH > 10.0 where the extent approached that observed in the presence of antimycin following a group of flashes. Reduction of cytochrome b‐561 in the presence of myxothiazol showed a lag of ~ 1 ms after the flash, followed by reduction with (t

~ 6 ms; by analogy with the similar kinetics of the quinol oxidase site, we suggest that the rate is determined by collision with the QH2 produced in the pool on flash excitation.
The primary structure of the gene encoding yeast ribosomal protein L 16
Authors: Leer Robert J. , van Raamsdonk-Duin Mary M.C. , Mager Willem H. , Planta Rudi J.
Published: July 2002

As part of our studies on the molecular basis for the coordinate expression of ribosomal protein genes in yeast we analyzed the primary structure of the gene encoding protein L16 of the large ribosomal subunit including the flanking sequences. L 16 turned out to be a ribosomal protein with a molecular mass of 22662 Da and a net charge of +12. Both the 5'‐ and the 3'‐end of the L16 mRNA were mapped by primer extension and S1 nuclease analysis. In the DNA regions flanking the coding sequence several conserved elements are present that may be involved in transcription initiation or termination.
Revised amino acid sequence of the B‐chain of ricin D due to loss of tryptophan in the cyanogen bromide cleavage
Authors: Araki Tomohiro , Funatsu Gunki
Published: July 2002

The complete amino acid sequence of the ricin D B‐chain was redetermined using the DABITC/PITC double‐coupling method. By comparing the presently established amino add sequence with the sequence reported earlier which was determined by the manual Edman degradation technique, it was found that the identification of 26 amino acid residues had to be revised. Furthermore, the ricin D B‐chain was shown to consist of 262 amino acid residues rather than 260 as previously described. In addition, based on the new amino acid sequence, the sequence homology between the N‐ and C‐terminal domain of this chain was found to be 31%.
A novel S = 3/2 EPR signal associated with native Fe‐proteins of nitrogenase
Authors: Hagen W.R. , Eady R.R. , Dunham W.R. , Haaker H.
Published: July 2002

In addition to their g = 1.94 EPR signal, nitrogenase Fe‐proteins from Azotobacter vinelandii, Azotobacter chroococcum and Klebsiella pneumoniae exhibit a weak EPR signal with g ≅5. Temperature dependence of the signal was consistent with an S = system with negative zero‐field splitting, d = −5 ± 0.7 cm−1. The m
s, = ± ground state doublet gives rise to a transition with g
eff = 5.90 and the transition within the excited m
s = ± doublet has a split g
eff = 4.8, 3.4. Quantitation gave 0.6 to 0.8 spin mol−1 which summed with the spin intensity of the S = g = 1.94 line to roughly 1 . MgATP and MgADP decreased the intensity of the s = signal with no concomitant changes in intensity of the s = signal.
Choice of peptide and peptide length for the generation of antibodies reactive with the intact protein
Authors: Welling Gjalt W. , Fries Hella
Published: July 2002

Antipeptide antibody
Ribonuclease
Secondary structure
Immunization
Potato virus X‐related single‐ and double‐stranded RNAs
Authors: Dolja Valerian V. , Grama Dmitrii P. , Morozov Sergei Yu. , Atabekov Joseph G.
Published: July 2002

Six species of 3′‐coterminal poly(A) ‐containing RNAs of subgenomic (sg) size have been found in plants infected with potato virus X (PVX): two major (0.9 kb — the coat protein mRNA, and 2.1 kb) and four minor (1.4, 1.8, 3.0 and 3.6 kb). The 5′‐end of the shortest sgRNA is located 26 nucleotides upstream of the initiating codon of the coat protein gene (812 nucleotides from the 3′‐terminal poly(A) tract of the PVX genomic RNA). Double‐stranded analogues have been found for most sgRNAs. The genomic‐size double‐stranded RNA (the replicative form) is shown to carry a poly(A)‐poly(U) hybrid of a predominant length of 150–250 bp on one end, and an unpaired G residue on the other (the 3′‐end of the negative chain). In contrast to this(—) the chains of double‐stranded 0.9 and 2.1 kbp sgRNAs lack the unpaired G and both end in C.
The transcription of B2 repeated sequences is regulated during the transition from quiescent to proliferative state in cultured rodent cells
Authors: Lania Luigi , Pannuti Antonio , La Mantia Girolama , Basilico Claudio
Published: July 2002

The RNA polymerase III‐dependent transcription of B2 repeated sequences has been monitored during the transition from the quiescent to proliferative state in cultured rodent cells and after polyomavirus‐induced transformation. The level of RNAs containing B2 sequences was found to be higher in both the proliferative state of normal cells and in polyomavirus‐transformed cells. In both systems, nuclear run‐off transcription assays indicated that high levels of B2 RNAs are due to an enhanced transcription rate. These results suggest the presence of a B2‐specific RNA pol III transcription factor(s) whose activity is sensitive to cell cycle progression and oncogenic transformation.
pH‐dependent fusion of reconstituted vesicular stomatitis virus envelopes with vero cells Measurement by dequenching of fluorescence
Authors: Paternostre Marie-Therese , Joel Lowy R. , Blumenthal Robert
Published: July 2002

Reconstituted vesicular stomatitis virus (VSV) envelopes were formed by solubilization of the viral envelope with Triton X‐100 followed by removal of detergent by direct addition of SM2 biobeads. We provide direct demonstration of fusion of reconstituted VSV with cells using fluorescent lipid and aqueous probes incorporated into the VSV virosomes during reconstitution. We show a direct comparison of the kinetics and pH profile of fusion with cells between reconstituted VSV and fluorescently labeled intact virus. With this preparation it is now possible to gain additional information about the role of cooperativity in viral protein‐mediated fusion, and to permit construction of efficient vehicles for delivery of drugs and other materials into cells.
Bacteriorhodopsin as a possible chloride pump
Authors: Dér András , Tóth-Boconádi Rudolf , Keszthelyi Lajos
Published: July 2002

Purple membranes oriented and immobilized in gel show charge transfer at pH 0.55 if the pH is set by HCl. Current appears as laser flash driven transient and also as continuous current by quasi‐continuous illumination. If the pH value 0.55 is set by H2SO4 continuous current is not observed. The results suggest that bacteriorhodopsin may pump chloride ions at low pH.
Benzo(a)pyrene induces nuclear‐DNA adducts in plant cell suspension culture
Authors: Rether B. , Pfohl-Leszkowicz A. , Guillemaut P. , Keith G.
Published: July 2002

Using a [32P] postlabelling technique, we found that plant cells (Echinacea purpurea) cultured in the presence of benzo(a)pyrene form nuclear DNA adducts.
Correlation between endotoxin‐neutralizing capacity of human plasma as tested by the limulus‐amebocyte‐lysate‐test and plasma protein levels
Authors: Berger D. , Schleich S. , Scidelmann M. , Beger H.G.
Published: July 2002

In the present study the endotoxin‐neutralizing capacity of human plasma obtained from healthy volunteers was determined by use of the limulus‐amebocyte‐lysate test. The extent of the endotoxin‐neutralizing capacity which showed a very broad variation was correlated to the plasma levels of some proteins which are believed to contribute to endotoxin transport and detoxification. The plasma levels of α2‐macroglobulin and transferrin, the major transport proteins, as well as the levels of IgG, IgA and IgM and apoprotein A were not significantly correlated to the neutralizing capacity. Only the apoprotein B, the major apoprotein of the low density lipoprotein fraction shows a significant correlation. Together with previously published results it should be concluded that low density lipoproteins are involved in the endogenous endotoxin‐neutralizing reaction occuring in human plasma, at least when high doses of endotoxin are administered. Nevertheless the endotoxinneutralizing reaction seems to be a very complex multistep process.
Ferredoxin‐dependent methane formation from acetate in cell extracts of Methanosarcina barkeri (strain MS)
Authors: Fischer R. , Thauer R.K.
Published: July 2002

Cell extracts of Methanosarcina barkeri grown on acetate catalyzed the conversion of acetyl‐CoA to CO2 and CH4 at a specific rate of 50 nmol·min−1·mg−1. When ferredoxin was removed from the extracts by DEAE‐Sephacel anion exchange chromatography, the extracts were inactive but full activity was restored upon addition of purified ferredoxin from M. barkeri or from Clostridiwn pasteurianum. The apparent Km for ferredoxin from M. barkeri was determined to be 2.5 μM. A ferredoxin dependence was also found for the formation of CO2, H2 and methylcoenzyme M from acetyl‐CoA, when methane formation was inhibited by bromoethanesulfonate. Reduction of methyl‐coenzyme M with H2 did not require ferredoxin. These and other data indicate that ferredoxin is involved as electron carrier in methanogenesis from acetate. Methanogenesis from acetyl‐CoA in cell extracts was not dependent on the membrane fraction, which contains the cytochromes.
Influenza virus A infection induces interleukin‐8 gene expression in human airway epitheial cells
Authors: Choi Augustine M.K. , Jacoby David B.
Published: July 2002

To determine the role of the airway epithelial cell in mediating virus‐induced inflammation, we infected primary cultures of human airway epithelial cells with human influenza type A/Port Chalmers/72 (H3N2). After two days, the medium was collected for measurement of the chemstactic cytokine interleukin‐8 by enzyme‐linked immunosorbent assay. The RNA was extracted from the cells for analysis of interleukin‐8 mRNA by Northern blot analysis. Interleukin‐8 production was more than doubled by viral infection, while interleukin‐8 mRNA was increased four‐folsd. Thus induction or interleukin‐8 gene expression in virus‐infected airway epithelium may be an important early step lending to virus‐induced airway inflammation.
RNA editing of atp6 transcripts from male‐sterile and normal cytoplasms of rapeseed (Brassica napus L.)
Authors: Handa Hirokazu , Nakajima Kousuke
Published: July 2002

The complete cDNA sequence corresponding to the rapeseed atp6 gene transcript (coding for subunit 6 of FO‐ATPase) has been determined by a method involving cDNA synthesis, using specific oligonucleotides as primers, followed by PCR amplification, cloning and sequencing of the amplification products. Only one modification, a C‐to‐U conversion, has been found when compared to the genomic mitochondrial DNA sequence. Comparison of the extent and frequency of RNA editing of the pol cytoplasmic male sterile (cms) atp6 transcript with those of normal atp6 transcript indicates that there is no variation between the editing status of the atp6 transcripts from pol cms and normal cytoplasms.
A peptide corresponding to a potential polyphosphoinositide binding site of phospholipase C‐β2 enhances its catalytic activity
Authors: Simoes Ana Paula , Schnabel Petra , Pipkom Rüdiger , Camps Montserrat , Gierschik Peter
Published: July 2002

A peptide corresponding to a basic consensus amino acid motif present in both actin‐binding proteins and phosphoinositide‐specific phospholipases C was synthesized and its effect on the activity of a recombinant phospholipase C‐β2 (PLCβ2) expressed in baculovirus‐infected insect cells was studied. The peptide markedly and specifically stimulated the activity of the enzyme. This stimulatory effect required a particular primary and/or secondary structure of the peptide and occurred without lowering the affinity of the enzyme for Ca2+. The function of the PLCβ2 segment corresponding to the peptide might be to bind and offer the substrate to the catalytic domain of this enzyme in a more favorable configuration or, alternatively, to interact with a hypothetical inhibitory constraint.
The CRE‐binding factor ATF‐2 facilitates the occupation of the CCAAT box in the fibronectin gene promoter
Authors: Srebrow Anabella , Muro Andres F. , Werbajh Santiago , Sharp Phillip A. , Kornblihtt Alberto R.
Published: July 2002

The cAMP response element (CRE) and the CCAAT box of the fibronectin gene promoter are separated by only twenty base pairs. A specific factor that binds the CRE interacts cooperatively with the protein which binds to the adjacent CCAAT box, stimulating transcription [1992, J. Biol. Chem. 267, 12767‐12774]. Here we show that the CRE factor is an heterodimer between a 43 kDa and the ‘73 kDa’ CRE‐binding proteins and we identify the latter as ATF‐2 (also named CRE‐BPI), a protein implicated in recruiting transcriptional activators to promoters, able to form heterodimers with Jun and for which a sequence‐deduced MW of 55 kDa had been previously reported.
Na+‐H+ exchange in sheep parotid endpieces
Authors: Poronnik P. , Young J.A. , Cook D.I.
Published: July 2002

We have used microspectrofluorimetry with the pH‐sensitive dye, BCECF, to examine the control of intracellular pH in the secretory endpieces of the sheep parotid gland. Unstimulated endpieces in HCO3
−‐free media have a cytosolic pH of 7.5 ± 0.03 (n = 69) which is maintained by a Na+‐dependent proton extrusion process that can be partially supported by Li+ but not by Cs+, and is not affected by changes in extracellular Cl−, HCO3
− or K+. It is not blocked by SITS or DIDS, which inhibit Na+‐(nHCO3
− co‐transport and Cl−1‐HCO3
− exchange, nor is it sensitive to the amiloride analogs, MIA and EIPA, which inhibit Na+‐H+ exchangers, although very high concentrations of amiloride itself (1 mmol/l) have a (probably non‐specific) inhibitory effect. It seems likely that sheep parotid secretory endpieces do contain a Na+‐H+ exchanger that drives secretion of a HCO3
−‐rich juice, and that its insensitivity to amiloride and its analogs explains why these drugs do not block fluid secretion by the intact sheep parotid gland.
Inhibition of protein prenylation by patulin
Authors: Miura Shinji , Hasumi Keiji , Endo Akira
Published: July 2002

The antibiotic patulin was found to inhibit protein prenylation in mouse FM3A cells. Thus, the agent reduced incorporation of [3H]mevalonate into proteins by 50% at a concentration of 7 μM. In a cell‐free assay, patulin inhibited rat brain farnesyl:protein transferase, one of the enzymes responsible for protein prenylation. The inhibition was 50% at a concentration of 290 μM.
Expression of the human D2S dopamine receptor in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe: A comparative study
Authors: Sander Peter , Grünewald Sylvia , Reiländer Helmut , Michel Hartmut
Published: July 2002

The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe were tested for heterologous expression of the human D2S dopamine receptor. The cDNA coding for the dopamine receptor was cloned into high copy number plasmids with inducible promoters. After transformation into the yeasts recombinant clones were examined for the presence of functional receptor by radioligand binding using the antagonist [3H]spiperone. Subsequent Western blot analysis of positive recombinants with an antiserum raised against a peptide from the third intracellular domain of the receptor protein revealed the production of a protein with an apparent molecular mass of 40 kDa in both yeasts. Membranes harvested from recombinant yeast clones exhibited saturable binding of the dopaminergic antagonist [3H]spiperone with K
d values of 1.3 nM in S. cerevisiae and 0.25 nM in S. pombe. The rank order of potencies for several dopaminergic ligands to displace specific [3H]spiperone binding to membranes were the same in both yeasts, whereas the affinities for ligands differed significantly.
Toxic principle of selva ant venom is a pore‐forming protein transformer
Authors: Arseniev A.S. , Pluzhnikov K.A. , Nolde D.E. , Sobol A.G. , Torgov M.Yu. , Sukhanov S.V. , Grishin E.V.
Published: July 2002

Ectatomin (Ea) is a newly isolated main toxic component of Ectatomma tuberculatum ant venom. Structural and electrophysiological studies were performed with purified Ea. The protein consists of two homologous polypeptide chains (37 and 34 residues) and forms a four α‐helix bundle in aqueous solution. On insertion into artificial bilayer membranes, two Ea molecules form an ion pore. Our results suggest that the ‘inside‐out’ mechanism of pore formation requires a significant movement of Ea helical parts. The pore formation in the cell membrane might well explain the toxic activity of Ea, not excluding at the same time its intracellular activities.
Positive and negative regulatory elements are involved in transcriptional control of the rat glucokinase gene in the insulin producing cell line HIT M2.2.2
Authors: Leibiger Ingo B. , Walther Reinhard , Pett Ute , Leibiger Barbara
Published: July 2002

Nested deletion mutants of the 5' flanking region of the β‐cell transcription unit of the rat glucokinase gene (rßGK) were fused to the CAT‐reporter gene. Transient expression studies in HIT M2.2.2 and BHK21 cells revealed a distal (upstream of −359) and a proximal promoter region (between −278/−49) harbouring positive and negative regulatory elements. DNaseI footprinting revealed three protected areas between nucleotides −190 and −60. DNA‐elements playing a crucial role in transcriptional control of the insulin genes (IEB‐ and CT‐motifs) have been detected within the proximal promoter region and contribute to β‐cell specific gene regulation. 3' deletion analysis revealed that DNA‐elements located downstream from transcription initiation sites (up to +123) contribute to transcriptional regulation.
Processing and transport of human small intestinal lactase‐phlorizin hydrolase (LPH)
Authors: Y. Naim Hassan
Published: July 2002

The effect of glycosylation on the intracellular transport of human intestinal lactase‐phlorizin hydrolase (LPH) was investigated by biosynthetic labeling of biopsy samples in the presence or absence of glycosidase inhibitors. In the presence of deoxynojirimycin (dNM) and deoxymannojirimycin (dMM), endo H sensitive LPH glycoforms of M
r = 135,000 in both cases were produced (LPH
dNM
and LPH
dMM
). The LPH glycoform generated in the presence of swainsonine had an apparent molecular mass of 141,000 (LPH
Swa
) and was partially sensitive to endo H. By contrast to unmodified mature LPH (LPHm
M
r = 160,000), these glycoforms are either not O‐glycosylated (LPH
dNM
and LPH
dMM
) or partially O‐glycosylated (LPH
Swa
) indicating that processing of N‐linked carbohydrates has direct effects on the O‐glycosylation of pro‐LPH. Analysis of transport kinetics of the various glycoforms strongly suggested that carbohydrate modification does not affect the transport of pro‐LPH from the cis‐Golgi to the cell surface, but could be rate limiting at the level of the ER.
Detection of two phospholipase A2(PLA2) activities in leaves of higher plant Vicia faba and comparison with mammalian PLA2's
Authors: Kyong Kim Dae , Lee Ho Joung , Lee Youngsook
Published: July 2002

Leaves of higher plant Vicia faba contains two Phospholipase A2 (PLA2) activities which are detected in cytosolic fractions. Based on a gel filtration column chromatography, two cytosolic PLA2 activities migrated with molecular masses of 70 kDa and 14 kDa. The first (70 kDa peak) was optimally active at pH 4.5 and was not dependent on [Ca2+ for its activity. In the presence of 5 mM CaCl2, ‘phospholipase B’ activity was shown in the 70 kDa peak. The second (14 kDa peak) was optimally active in the pH range 9–10 and required millimolar concentrations of calcium for optimal activity. The two activities were not inhibited by dithiothreitol. Neither anti‐pancreatic PLA2 antiserum nor anti‐(pig spleen 100 kDa cytosolic PLA2) antiserum immunoprecipitated any activity of the two plant PLA2's. The present results indicate that at least the 14 kDa form of the two PLA2 enzymes detected in leaves of higher plants is biochemically and immunochemically different from the well characterized Ca2+‐dependent mammalian PLA2's.
REGULATED EXPRESSION OF CHONDROITIN SULFATES AT SITES OF EPITHELIAL—MESENCHYMAL INTERACTION: SPATIO‐TEMPORAL PATTERNING IDENTIFIED WITH ANTI‐CHONDROITIN SULFATE MONOCLONAL ANTIBODIES
Authors: Sorrell J. Michael , Carrino David A. , Caplan Arnold I.
Published: July 2002

Chondroitin sulfate proteoglycans, cell surface and extracellular matrix molecules in both neural and non‐neural tissues, are highly regulated during normal development. Entire proteoglycan molecules may be either up‐regulated or down‐regulated, or only the chondroitin sulfate glycosaminoglycan portions of these molecules may be modified. Subtle changes in the chemistries of chondroitin sulfate chains can now be identified through the use of a panel of anti‐chondroitin sulfate monoclonal antibodies. Each of these antibodies recognizes specific chemical structures which are non‐randomly dispersed along the lengths of chondroitin sulfate chains. The location of individual epitopes within defined domains in these chains is demonstrated through controlled treatments of aggrecan with chondroitinase ABC, whereby portions of these chains are removed from the non‐reducing terminal ends and where the remainder of the chains remains covalently attached to the core protein. In these situations, some epitopes, such as those recognized by antibodies CS‐56 and 6C3, can be removed without loss of other epitopes, such as that recognized by antibody 4C3. The independent expression of individual epitopes is demonstrated by immunocytochemical analyses of developing skin appendages in embryonic chicks and fetal humans. These are sites where highly patterned morphogenetic movements result from epithelial‐mesenchymal interactions. In both chicks and humans, some epitopes are constitutively expressed while others are strictly regulated in the mesenchymal portions of the developing skin appendages. These data strongly suggest that chondroitin sulfate proteoglycans, including their chondroitin sulfate chains, have important roles in regulating these epithelial—mesenchymal interactions. Furthermore, these data underscore the significance of the aforementioned observation that individual epitopes are located in specific domains within chondroitin sulfate chains. The highly organized expression of chondroitin sulfate proteoglycans in the development of the central nervous system strongly argues for a similar role for these molecules in the organs that comprise this system.
Analysis of the domain requirement in Gas1 growth suppressing activity
Authors: Ruaro Maria Elisabetta , Stebel Marco , Vatta Paolo , Marzinotto Stefania , Schneider Claudio
Published: July 2002

The product of the growth arrest specific gene, gas1, is a membrane‐associated protein which activates a p53‐dependent growth suppression signalling pathway. We have shown that Gas1 is linked to the plasma membrane through a glycosyl‐phosphatidylinositol (GPI) anchor. Several GPI‐anchored protein have been identified as part of receptor complexes either as co‐receptors or as membrane bound ligands. In this report, we characterize the Gas1 domains required for its growth suppression function and demonstrate the dispensability of Gas1 GPI anchor.
Genomic Exploration of the Hemiascomycetous Yeasts: 16. Candida tropicalis
Authors: Blandin Gaëlle , Ozier-Kalogeropoulos Odile , Wincker Patrick , Artiguenave François , Dujon Bernard
Published: July 2002

The genome of the diploid hemiascomycetous yeast Candida tropicalis, an opportunistic human pathogen and an important organism for industrial applications, was explored by the analysis of 2541 Random Sequenced Tags (RSTs) covering about 20% of its genome. Comparison of these sequences with Saccharomyces cerevisiae and other species permitted the identification and the analysis of a total of more than 1000 novel genetic elements of C. tropicalis. Moreover, the present study confirms that in C. tropicalis, the rare CUG codon is read as a serine and not a leucine. The sequences have been deposited at EMBL with the accession numbers AL438875–AL441602.
Tyrosine 331 and phenylalanine 334 in Clostridium perfringens α‐toxin are essential for cytotoxic activity
Authors: Jepson Marie , Bullifent Helen L. , Crane Dennis , Flores-Diaz Marietta , Alape-Giron Alberto , Jayasekeera Pramukh , Lingard Bryan , Moss David , Titball Richard W.
Published: July 2002

Differences in the biological properties of the Clostridium perfringens phospholipase C (α‐toxin) and the C. bifermentans phospholipase C (Cbp) have been attributed to differences in their carboxy‐terminal domains. Three residues in the carboxy‐terminal domain of α‐toxin, which have been proposed to play a role in membrane recognition (D269, Y331 and F334), are not conserved in Cbp (Y, L and I respectively). We have characterised D269Y, Y331L and F334I variant forms of α‐toxin. Variant D269Y had reduced phospholipase C activity towards aggregated egg yolk phospholipid but increased haemolytic and cytotoxic activity. Variants Y331L and F334I showed a reduction in phospholipase C, haemolytic and cytotoxic activities indicating that these substitutions contribute to the reduced haemolytic and cytotoxic activity of Cbp.
Evidence for the putative docking/fusion complex of exocytosis in parotid acinar cells
Authors: Takuma Taishin , Tagaya Mitsuo , Ichida Tokuro
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Tyrphostin AG 494 blocks Cdk2 activation
Authors: Osherov Nir , Levitzki Alexander
Published: July 2002

We have previously shown that the EGFR kinase selective tyrphostin AG 494 fails to inhibit EGFR kinase in intact cells. Yet, AG 494 proved to inhibit EGF‐ or serum‐induced cell proliferation (Osherov et al., J. Biol. Chem. 268 (1993) 11134–11142). In this preliminary communication we show that AG 494 as well as its close analogs AG 490 and AG 555 block Cdk2 activation. In contrast, AG 1478, a more selective EGFR kinase blocker which is also active as EGFR kinase blocker in intact cells, fails to do so. AG 494 exerts its full inhibitory activity on Cdk2 activation even when added 20 h subsequent to EGF addition when Cdk2 activation is maximal. The inhibitory activity on Cdk2 activation parallels its DNA synthesis inhibitory activity, strongly suggesting that its target is one of the molecular mechanisms involved in Cdk2 activation. AG 494 and its analogs may become useful lead compounds for the development of drugs aimed at the cell cycle machinery.
Overexpression of thioredoxin in Fanconi anemia fibroblasts prevents the cytotoxic and DNA damaging effect of mitomycin C and diepoxybutane
Authors: Ruppitsch Werner , Meißlitzer Claudia , Hirsch-Kauffmann Monica , Schweiger Manfred
Published: July 2002

Adult T cell leukemia derived factor (ADF)/thioredoxin (Trx) is known to be an important intracellular antioxidant involved in a number of redox reactions such as ribonucleotide reductase (RNR) as well as of tyrosinase. Since RNR is a key enzyme of nucleotide metabolism and DNA synthesis, a reduced Trx level would result in reduced enzymatic activity and cause DNA damage. Furthermore, Trx is considered to be an effective regulator of redox sensitive gene expression. The role of Trx in nucleotide metabolism and gene expression may be an explanation for increased chromosomal instability as well as hypersensitivity towards oxygen, ROI and ROI generating agents. The activity of tyrosinase, the key enzyme of melanin biosynthesis, is influenced by the thioredoxin level and by superoxide radicals. Low thioredoxin levels and high superoxide concentrations activate tyrosinase causing hyperpigmentation of the skin. In addition to the observed high superoxide concentration in Fanconi anemia (FA) patients, a low thioredoxin level might be responsible for the hyperpigmentation (café‐au‐lait spots) in this disease. We observed that overexpression of the thioredoxin cDNA in FA fibroblasts completely abolished the DNA damaging effects of mitomycin C and diepoxybutane and inhibited the constitutive activity of the nuclear factor κB (NF‐κB) in SV40 transformed FA fibroblasts. However, spontaneous chromosomal breakage was not affected.
A deep brain photoreceptive molecule in the toad hypothalamus
Authors: Yoshikawa Tomoko , Okano Toshiyuki , Oishi Tadashi , Yoshitaka Fukada
Published: July 2002

We have isolated a cDNA clone encoding a deep brain photoreceptive molecule from the hypothalamic cDNA library of the toad, Bufo japonicus. The deduced amino acid sequence showed the highest similarity to that of pinopsin (75–76%) among vertebrate retinal opsins, indicating the expression of toad pinopsin in the deep brain. Antibodies raised against the C‐terminal tail of toad pinopsin stained cell bodies and the knob‐like structures of the cerebrospinal fluid‐contacting neurons in the anterior preoptic nucleus. This region is known to play an important role in breeding behavior, suggesting that toad pinopsin acts as a photosensor for the photoperiodic gonadal response.
The carboxy‐terminal region of adenovirus E1A activates transcription through targeting of a C‐terminal binding protein‐histone deacetylase complex
Authors: Sundqvist Anders , Sollerbrant Kerstin , Svensson Catharina
Published: July 2002

Binding of the C‐terminal binding protein, CtBP, to the adenovirus E1A moiety of a Gal4‐E1A fusion protein abolishes conserved region (CR) 1‐dependent transcription activation. In contrast, a non‐promoter targeted E1A peptide, capable of binding CtBP, can induce transcription from the proliferating cell nuclear antigen (PCNA) promoter. CtBP is shown here to bind the histone deacetylase HDAC1, suggesting that a promoter targeted CtBP‐HDAC1 complex can silence transcription from the PCNA promoter through a deacetylation mechanism. Expression of the CtBP binding domain of E1A is sufficient to alleviate repression, possibly due to the displacement of the CtBP‐HDAC1 complex from the promoter.
Mitochondrial Hsp70 cannot replace BiP in driving protein translocation into the yeast endoplasmic reticulum
Authors: Brodsky Jeffrey L , Bäuerle Marc , Horst Martin , McClellan Amie J
Published: July 2002

To determine whether mitochondrial hsp70 (mHsp70) could substitute for the endoplasmic retuculum (ER) Hsp70 (BiP) during protein translocation, we assembled ER‐derived reconstituted proteoliposomes supplemented with either protein. We found that only BiP restored translocation in kar2 mutant vesicles and stimulated translocation ∼3‐fold in wild type proteoliposomes. mHsp70 associated poorly with both a BiP binding (DnaJ) domain of Sec63p and an ER precursor, and its ATPase activity was poorly enhanced upon incubation with the DnaJ domain. In contrast, BiP bound to the Sec63p‐DnaJ domain in an ATP‐dependent manner and its ATPase activity was stimulated significantly by this polypeptide. We conclude that mHsp70 is unable to support protein translocation into the ER because it fails to associate productively with Sec63p and a precursor.
Cloning of the V‐ATPase subunit G in plant: functional expression and sub‐cellular localization1
Authors: Rouquié David , Tournaire-Roux Colette , Szponarski Wojciech , Rossignol Michel , Doumas Patrick
Published: July 2002

A 13‐kDa tobacco plasma membrane protein was isolated from two‐dimensional electrophoresis gels. After microsequencing, RT‐PCR techniques and cDNA library screening allowed for the cloning of two cDNAs. These cDNAs encoded for the subunit G of the vacuolar H+‐ATPase, the first one identified in plants. Analysis of mRNA distribution showed a maximum level in the leaves and in the stem of the apical part of the tobacco plant. Heterologous functional complementation of the yeast mutant (Δvma10::URA3) was achieved with the two cDNAs. After fractionation of microsomal membranes on linear sucrose gradient, Western blots were performed using antibodies against recombinant protein and three peaks were identified: one which comigrated with the tonoplast marker and the others at slightly higher density corresponding to endoplasmic reticulum and to plasma membrane fractions.
Cyc2p is required for maintaining ionic stability and efficient cytochrome c import and mitochondrial function in Saccharomyces cerevisiae
Authors: Pearce David A. , Cardillo Thomas S. , Sherman Fred
Published: July 2002

A previous study demonstrated that Cyc2p from Saccharomyces cerevisiae is a mitochondrial protein and that cyc2 mutants contained only approximately 20% of the normal levels of cytochrome c due to a partial deficiency in mitochondrial import of apo‐cytochrome c. We report herein that deletion of the entire gene results in defective mitochondrial function, as revealed by diminished growth on media containing non‐fermentable carbon sources. This defect is exacerbated in hyper‐ionic KCl media and at higher incubation temperatures, but is suppressed on media containing sorbitol, a non‐ionic compound. We suggest that Cyc2p serves to maintain the osmotic stability of mitochondria, and its defect is exacerbated by KCl.
Role of the third intracellular loop of the Angiotensin II receptor subtype AT2 in ligand‐receptor interaction
Authors: Dittus Jason , Cooper Shannon , Obermeir Gerald , Pulakat Lakshmidevi
Published: July 2002

Angiotensin II (Ang II) receptor subtypes AT1 and AT2 share 34% overall homology, but the least homology is in their third intracellular loop (3rd ICL). In an attempt to elucidate the role of the 3rd ICL in determining the similarities and differences in the functions of the AT1 and the AT2 receptors, we generated a chimeric receptor in which the 3rd ICL of the AT2 receptor was replaced with that of the AT1 receptor. Ligand‐binding properties and signaling properties of this receptor were assayed by expressing this receptor in Xenopus oocytes. Ligand‐binding studies using [125I‐Sar1‐Ile8] Ang II, a peptidic ligand that binds both the AT1 and the AT2 receptor subtypes, and 125I‐CGP42112A, a peptidic ligand that is specific for the AT2 receptor, showed that the chimeric receptor has lost affinity to both ligands. However, IP3 levels of the oocytes expressing the chimeric receptor were comparable to the IP3 levels of the oocytes expressing the AT1 receptor, suggesting that the chimeric receptors could couple to phospholipase C pathway in response to Ang II. We have shown previously that the nature of the amino acid present in the position 215 located in the fifth transmembrane domain (TMD) of the AT2 receptor plays an important role in determining its affinity to different ligands. Our results from the ligand‐binding studies of the chimeric receptor further support the idea that the structural organization of the region spanning the 5th TMD and the 3rd ICL of the AT2 receptor has an important role in determining the ligand‐binding properties of this receptor.
Application of metabolic control analysis to the study of toxic effects of copper in muscle glycolysis
Authors: Jannaschk Dietmar , Burgos Montse , Centerlles Josep J , Ovadi Judit , Cascante Marta
Published: July 2002

Experimental and model studies have been performed to characterise the effects of Cu2+ on the activities of individual glycolytic enzymes and on the flux and internal metabolite concentrations of the upper part of glycolysis in mouse muscle extracts. Cu2+ significantly inhibited the triosephosphate production from glucose with an IC50 of about 6.0 μM. At a similar extension Cu2+ inhibited hexokinase and phosphofructokinase, with an IC50 of 6.2 μM and 6.4 μM respectively, whereas the effects on the activities of aldolase, phosphoglucose isomerase and the internal metabolite levels were not significant. Flux control coefficients and flux response coefficients were determined in the presence of copper concentrations between 0 and 10 μM. The same values of flux control coefficients for hexokinase and for phosphofructokinase (0.8 and 0.2 respectively) were found in absence and in presence of copper. At Cu2+ equal to the flux IC50, the response coefficient was −1. The elasticity coefficients for hexokinase and phosphofructokinase at Cu2+ equal to the IC50 were also −1. A mathematical model was used to analyze the effect of copper on glycolysis under different conditions using experimental kinetic parameters and rate equations for enzymatic reactions of the upper part of glycolysis.
Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution
Authors: Doi Nobuhide , Yanagawa Hiroshi
Published: July 2002

Protein‐engineering techniques have been adapted for the molecular design of biosensors that combine a molecular‐recognition site with a signal‐transduction function. The optical signal‐transduction mechanism of green fluorescent protein (GFP) is most attractive, but hard to combine with a ligand‐binding site. Here we describe a general method of creating entirely new molecular‐recognition sites on GFPs. At the first step, a protein domain containing a desired molecular‐binding site is inserted into a surface loop of GFP. Next, the insertional fusion protein is randomly mutated, and new allosteric proteins that undergo changes in fluorescence upon binding of target molecules are selected from the random library. We have tested this methodology by using TEM1 β‐lactamase and its inhibitory protein as our model protein‐ligand system. ‘Allosteric GFP biosensors’ constructed by this method may be used in a wide range of applications including biochemistry and cell biology.
Evidence for the presence of several phosphodiesterase isoforms in brown adipose tissue of Zucker rats: modulation of PDE2 by the fa gene expression
Authors: Coudray Carole , Charon Christine , Komas Narcisse , Mory Gérard , Diot-Dupuy Francine , Manganiello Vincent , Ferre Pascal , Bazin Raymond
Published: July 2002

The present study was undertaken to characterise the phosphodiesterases (PDEs) present in brown adipose tissue (BAT) of Zucker rat pups and to determine whether the capacity for degradation of cyclic nucleotides was affected by the fatty genotype. Regardless of the genotype, PDE2–4 contributed to total PDE activity, the PDE3 activity equalling the sum of PDE2 and 4 activities. In fa/fa compared to Fa/fa rats, (a) PDE2 activity was significantly increased, (b) Western blot analysis of PDE2 revealed two signals at 71 and 105 kDa, with changes in protein being in good parallelism with changes in activity, (c) the PDE2 mRNA concentration was also significantly increased. In good agreement, the cGMP concentration was decreased in BAT from fa/fa pups.
Spatial and temporal regulation of protein expression by bldA within a Streptomyces lividans colony
Authors: Kataoka Masakazu , Kosono Saori , Tsujimoto Gozoh
Published: July 2002

The bldA gene encodes the only tRNA for the UUA codon that, although dispensable in genes important for primary vegetative growth of Streptomyces spp., is important in genes that serve a regulatory purpose in the differentiation. To investigate this role further, the spatial and temporal expression profiles of the bldA‐regulated and unregulated genes within a Streptomyces colony were examined using modified genes for the green fluorescent protein (gfp) as an expression‐tag. A comparative study, based on computer‐assisted quantitative analysis of the GFP fluorescence, revealed that the presence of TTA codons in gfp results in a temporal delay of translation and, consequently, changed the spatial pattern of the GFP expression within a colony, especially during early differentiation. The delay of GFP expression was undetectable at 60 h post‐inoculation. These results provide the first extensive evidence that the bldA does indeed play a significant regulatory role during colony differentiation.