bluuebunny/crossref_metadata_2025 huggingface dataset:
Screening of 49 antibiotic residues in aquatic products using modified QuEChERS sample preparation and UPLC-QToFMS analysis
Authors: Gao Yao , Zhang Tianwen , Huang Shirong , Lin Xinxin , Gong Sisi , Chen Qiuhua , Huang Dongren , Chen Min
Published: February 2021

A precise analytical method was established for rapid screening of 49 antibiotic residues in aquatic products by ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QToFMS). The quick, easy, cheap, effective, rugged and safe (QuEChERS) process was refined for effective sample preparation. The homogenized samples of aquatic products were extracted with 3% acetic acid in acetonitrile, salted out with anhydrous magnesium sulfate and sodium chloride, and cleaned up by octadecylsilane (C18) and primary-secondary amine (PSA) powder. Then, the purified samples were separated on a BEH C18 column using 0.1% formic acid and methanol as mobile phases by gradient elution, detected by MS under positive Electron Spray Ionization (ESI+) mode. The linear range of matrix-matched calibration curve was 1–100 μg/L for each compound with the correlation coefficients in the range of 0.9851–0.9999. The recoveries of target antibiotics at the different spiked levels ranged from 60.2% to 117.9% except for lincomycin hydrochloride, whereas relative standard deviations (RSDs) were between 1.6% and 14.0% except for sulfaguanidine in grass Carp, Penaeus vannamei and Scylla serrata matrices. The limits of detection (LODs) (S/N = 3) for the analytes were 0.05–2.40 μg/kg, 0.08–2.00 μg/kg and 0.10–2.27 μg/kg and the limits of quantification (LOQs) (S/N = 10) were 0.16–8.00 μg/kg, 0.25–6.66 μg/kg and 0.32–7.56 μg/kg in grass Carp, Penaeus vannamei and Scylla serrata, respectively. The method was successfully applied to grass Carp, Penaeus vannamei and Scylla serrata, demonstrating its ability for the determination of multi-categories antibiotic residues in aquatic products.
Efficient steganalysis using convolutional auto encoder network to ensure original image quality
Authors: Ayaluri Mallikarjuna Reddy , K. Sudheer Reddy , Konda Srinivasa Reddy , Chidirala Sudharshan Reddy
Published: February 2021

Steganalysis is the process of analyzing and predicting the presence of hidden information in images. Steganalysis would be most useful to predict whether the received images contain useful information. However, it is more difficult to predict the hidden information in images which is computationally difficult. In the existing research method, this is resolved by introducing the deep learning approach which attempts to perform steganalysis tasks in effectively. However, this research method does not concentrate the noises present in the images. It might increase the computational overhead where the error cost adjustment would require more iteration. This is resolved in the proposed research technique by introducing the novel research method called Non-Gaussian Noise Aware Auto Encoder Convolutional Neural Network (NGN-AEDNN). Classification technique provides a more flexible way for steganalysis where the multiple features present in the environment would lead to an inaccurate prediction rate. Here, learning accuracy is improved by introducing noise removal techniques before performing a learning task. Non-Gaussian Noise Removal technique is utilized to remove the noises before learning. Also, Gaussian noise removal is applied at every iteration of the neural network to adjust the error rate without the involvement of noisy features. This proposed work can ensure efficient steganalysis by accurate learning task. Matlab has been employed to implement the method by performing simulations from which it is proved that the proposed research technique NGN-AEDNN can ensure the efficient steganalysis outcome with the reduced computational overhead when compared with the existing methods.
An adaptive weighting mechanism for Reynolds rules-based flocking control scheme
Authors: Hoang Duc N. M. , Tran Duc M. , Tran Thanh-Sang , Pham Hoang-Anh
Published: February 2021

Cooperative navigation for fleets of robots conventionally adopts algorithms based on Reynolds's flocking rules, which usually use a weighted sum of vectors for calculating the velocity from behavioral velocity vectors with corresponding fixed weights. Although optimal values of the weighting coefficients giving good performance can be found through many experiments for each particular scenario, the overall performance could not be guaranteed due to unexpected conditions not covered in experiments. This paper proposes a novel control scheme for a swarm of Unmanned Aerial Vehicles (UAVs) that also employs the original Reynolds rules but adopts an adaptive weight allocation mechanism based on the current context than being fixed at the beginning. The simulation results show that our proposed scheme has better performance than the conventional Reynolds-based ones in terms of the flock compactness and the reduction in the number of crashed swarm members due to collisions. The analytical results of behavioral rules’ impact also validate the proposed weighting mechanism's effectiveness leading to improved performance.
A comparison of fourteen fully characterized mammalian-associated Campylobacter fetus isolates suggests that loss of defense mechanisms contribute to high genomic plasticity and subspecies evolution
Authors: Nadin-Davis Susan A. , Chmara John , Carrillo Catherine D. , Amoako Kingsley , Goji Noriko , Duceppe Marc-Olivier , Devenish John
Published: February 2021

Campylobacter fetus is currently classified into three main subspecies, but only two of these, C. fetus subspecies fetus and C. fetus subsp. venerealis originate principally from ruminants where they inhabit different niches and cause distinct pathogenicity. Their importance as pathogens in international trade and reporting is also different yet the criteria defining these properties have never been fully substantiated nor understood. The situation is further compromised because the ability to differentiate between these two closely related C. fetus subspecies has traditionally been performed by phenotypic characterisation of isolates, methods which are limited in scope, time-consuming, tedious, and often yield inconsistent results, thereby leading to isolate misidentification. The development of robust genetic markers that could enable rapid discrimination between C. fetus subsp. fetus and subsp. venerealis has also been challenging due to limited differences in the gene complement of their genomes, high levels of sequence repetition, the small number of closed genome sequences available and the lack of standardisation of the discriminatory biochemical tests employed for comparative purposes. To yield a better understanding of the genomic differences that define these C. fetus strains, seven isolates were exhaustively characterised phenotypically and genetically and compared with seven previously well characterised isolates. Analysis of these 14 C. fetus samples clearly illustrated that adaption by C. fetus subsp. venerealis to the bovine reproductive tract correlated with increasing genome length and plasticity due to the acquisition and propagation of several mobile elements including prophages, transposons and plasmids harbouring virulence factors. Significant differences in the repertoire of the CRISPR (clustered regularly interspersed short palindromic repeats)-cas system of all C. fetus strains was also found. We therefore suggest that a deficiency in this adaptive immune system may have permitted the emergence of extensive genome plasticity and led to changes in host tropism through gene disruption and/or changes in gene expression. Notable differences in the sub-species complement of DNA adenine methylase genes may also have an impact. These data will facilitate future studies to better understand the precise genetic differences that underlie the phenotypic and virulence differences between these animal pathogens and may identify additional markers useful for diagnosis and sub-typing.
Extended Hybrid Genetic Algorithm for Solving Job Shop Scheduling Problem
Authors: Eshim Sumaia E. , Hamed Mohammed M.
Published: February 2021

In this paper, a hybrid genetic algorithm (HGA) to solve the job shop scheduling problem (JSSP) to minimize the makespan is presented. In the HGA, heuristic rules are integrated with genetic algorithm (GA) to improve the solution quality. The purpose of this research is to investigate from the convergence of a hybrid algorithm in achieving a good solution for new benchmark problems with different sizes. The results are compared with other approaches. Computational results show that a hybrid algorithm is capable to achieve good solution for different size problems.
Smart Traffic Management for Collision Avoidance Using V2V
Authors: Naren Thiruvalar V. , Vimal E.
Published: February 2021

The main objective of this project is to connect the vehicles together and avoid accidents by using V2V Communication. The vehicles are to be connected together by means of DSRC algorithm which is used for transceiving alert messages among the connected vehicles, in case of any emergency situation such as accidents. The Vehicle-to-Vehicle (V2V) and Vehicle-to- Infrastructure (V2I) technologies are specific cases of IoT and key enablers for Intelligent Transportation Systems (ITS). V2V and V2I have been widely used to solve different problems associated with transportation in cities, in which the most important is traffic congestion. A high percentage of congestion is usually presented by the inappropriate use of resources in vehicular infrastructure. In addition, the integration of traffic congestion in decision making for vehicular traffic is a challenge due to its high dynamic behaviour. An increase in the infrastructure growth is a possible solution but turns out to be costly in terms of both time and effort. Various applications that target transport efficiency could make use of the vast information collected by vehicles: safety, traffic management, pollution monitoring, tourist information, etc.
Calcium ions can be substituted for the 24‐kDa polypeptide in photosynthetic oxygen evolution
Authors: Miyao Mitsue , Murata Norio
Published: July 2002

Photosystem II particles were prepared from spinach chloroplasts with Triton X‐100, and treated with 1.0 M NaCl to remove polypeptides of 24 kDa and 18 kDa and to reduce the photosynthetic oxygen‐evolution activity by about half. Oxygen‐evolution activity was restored almost to the original level with 10 mM Ca2+, in a similar manner to the rebinding of 24‐kDa polypeptide. Other cations such as magnesium, sodium and manganese ions could not restore any oxygen‐evolution activity. These observations, together with a kinetic analysis, suggest that Ca2+ can be substituted for the 24‐kDa polypeptide in photosynthetic oxygen evolution in Photosystem II particles.
Sidedness of plant plasma membrane vesicles purified by partitioning in aqueous two‐phase systems
Authors: Larsson C. , Kjellbom P. , Widell S. , Lundborg T.
Published: July 2002

Preparations of plant plasma membrane vesicles were obtained by partition in dextran‐polyethylene glycol two‐phase systems. By this procedure particles are separated according to their surface properties, and an iso‐osmotic environment is maintained throughout. The vesicles thus produced are right‐side‐out and sealed, as measured by enzyme [(K+ + Mg2+)‐ATPase and glucan synthetase II] latency on addition of Triton X‐100.
Glycerol stabilizes oxygen evolution and maintains binding of a 9 kDa polypeptide in photosystem II particles from the cyanobacterium, Phormidium laminosum
Authors: Stewart Alison C. , Siczkowski Martin , Ljungberg Ulf
Published: July 2002

High concentrations of glycerol (⩾20% ) stabilize oxygen evolution in photosystem (PS) II particles from the thermophilic cyanobacterium, Phormidium laminosum. Treating PS II particles with lower glycerol concentrations inhibits activity and, in parallel, detaches a 9 kDa polypeptide from PS II. These results suggest that the 9 kDa polypeptide is essential for PS II activity, and that it is bound to PS II primarily by hydrophobic interactions that are strengthened in vitro by glycerol. The 9 kDa polypeptide is distinct from cytochrome b‐559 and does not appear to be associated with manganese or with the calcium or chloride requirements for oxygen evolution.
Binding sites for calcium, lipid and p11 on p36, the substrate of retroviral tyrosine‐specific protein kinases
Authors: Johnsson Nils , Vandekerckhove Joel , Van Damme Jozef , Weber Klaus
Published: July 2002

Biochemical and partial sequence data reveal the two‐domain structure of p36. A loose structure of some 30 residues at the amino‐terminus contains the phosphorylatable tyrosine and the binding site for the p11 regulatory chain. The following p33 domain retains the lipid‐binding site as well as the Ca2+ site which influences the spectral properties of the single tryptophan and one tyrosine. The combined sequence data covering about 25% of the molecule identify p36 as a unique polypeptide.
ΔpH‐dependent chlorophyll fluorescence quenching indicating a mechanism of protection against photoinhibition of chloroplasts
Authors: Krause G.Heinrich , Behrend Ursula
Published: July 2002

Intact isolated spinach chloroplasts were subjected to photoinhibitory conditions (high light and lack of CO2). Photoinhibition of the electron transport system was considerably diminished when the chloroplasts were in a low‐fluorescent state related to a high proton gradient across the thylakoid membranes, as compared to a high‐fluorescent state in which ΔpH‐dependent fluorescence quenching was abolished by addition of uncouplers. The hypothesis is discussed that in chloroplasts exposed to excess light, photoinhibition is partly prevented by increased thermal dissipation of excitation energy, as expressed by ΔpH‐dependent (‘energy‐dependent’) chlorophyll a fluorescence quenching.
A method for removal of trace iron contamination from biological buffers
Authors: Gutteridge John M.C.
Published: July 2002

Laboratory chemicals and reagents normally contain trace amounts of iron salts sufficient to catalyse free radical reactions. This iron contamination can be removed from buffers and reagents using a dialysis sac containing a high‐affinity iron‐binding protein like conalbumin or transferrin without altering the pH value of the fluid.
Identification of the phosphorylation site of an 8.3 kDa protein from photosystem II of spinach
Authors: Michel H.P. , Bennett J.
Published: July 2002

The four principal phosphoproteins of PS II cores (8.3, 32, 34 and 44 kDa) give rise to distinct tryptic phosphopeptides which have been purified by affinity chromatography on Fe3+‐chelating Sepharose and reverse‐phase HPLC. The tryptic phosphopeptide derived from the 8.3 kDa protein has the sequence NH2‐Ala‐Thr‐Gln‐Thr‐Val‐Glu‐Ser‐Ser‐Ser‐Arg. It corresponds to the N‐terminus of the chloroplast psbH gene product, except for the loss of the initiating N‐formylmethionine. The peptide is phosphorylated on the first threonyl residue. Differences between the phosphorylation sites of the 8.3 kDa protein and LHC II are consistent with the hypothesis that thylakoids contain two distinct redox‐controlled protein kinases differing in substrate specificity.
Aromatic ring cleavage of 4,6‐di(tert‐butyl)guaiacol, a phenolic lignin model compound, by laccase of Coriolus versicolor
Authors: Kawai Shingo , Umezawa Toshiaki , Shimada Mikio , Higuchi Takayoshi
Published: July 2002

It was found that 2,4‐di(tert‐butyl)‐4‐(methoxycarbonylmethyl)‐2‐buten‐4‐olide (II) was formed as an aromatic ring cleavage product of a phenolic lignin model compound, 4,6‐di(tert‐butyl)guaiacol (I), by laccase of Coriolus versicolor. Based on isotopic experiments with 18O2 H2
18O, the mechanism of formation of II from I is discussed.
Low frequency electric and magnetic fields have different effects on the cell surface
Authors: Marron Michael T. , Goodman Eugene M. , Sharpe Paul T. , Greenebaum Ben
Published: July 2002

There is a considerable controversy over the nature of weak electromagnetic‐field effects in living organisms. Part of the controversy can be traced to a lack of understanding of whether electric or magnetic fields are involved in producing bioeffects. We find that both 60 Hz electric and magnetic fields alter the cell surface of Physarum polycephalum. Exposure to electric fields increases the negative charge on the cell surface while magnetic‐field exposure decreases the hydrophobic character of the surface. These effects appear to be additive and independent of the waveform of the applied fields.
Further analysis of cDNA clones for maize phosphoenolpyruvate carboxylase involved in C4 photosynthesis Nucleotide sequence of entire open reading frame and evidence for polyadenylation of mRNA at multiple sites in vivo
Authors: Yanagisawa Shuichi , Izui Katsura , Yamaguchi Yasunori , Shigesada Katsuya , Katsuki Hirohiko
Published: July 2002

Four clones of cDNA for phosphoenolpyruvate carboxylase [EC 4.1.1.31] were obtained from a maize green leaf cDNA library by colony hybridization. The largest cDNA was of full‐length (3335 nucleotides), being 243 nucleotides longer than the cDNA cloned previously [(1986) Nucleic Acids Res. 14, 1615–1628]. Alignment of the sequence for the N‐terminal coding region found in two of the four clones with the sequence reported previously, established the sequence of the entire coding region for the enzyme. The sequencing of 3′‐untranslated region of the clones revealed that the poly(A) tract is attached at multiple sites in vivo.
Proteins from the prokaryotic nucleoid Interaction of nucleic acids with the 15 kDa Escherichia coli histone‐like protein H‐NS
Authors: Friedrich K. , Gualerzi C.O. , Lammi M. , Losso M.A. , Pon C.L.
Published: July 2002

The interaction between nucleic acids and Escherichia coli H‐NS, an abundant 15 kDa histone‐like protein, has been studied by affinity chromatography, nitrocellulose filtration and fluorescence spectroscopy. Intrinsic fluorescence studies showed that the single Trp residue of H‐NS (position 108) has a restricted mobility and is located within an hydrophobic region inaccessible to both anionic and cationic quenchers. Binding of H‐NS to nucleic acids, however, results in a change of the microenvironment of the Trp residue and fluorescence quenching; from the titration curves obtained with addition of increasing amounts of poly(dA)‐poly(dT) and poly(dC)‐poly(dG) it can be estimated that an H‐NS dimer in 1.5 × SSC binds DNA with an apparent K
a−1.1 × 104 M−1· bp−1. H‐NS binds to double‐stranded DNA with a higher affinity than the more abundant histone‐like protein NS(HU) and, unlike NS, prefers double‐stranded to single‐stranded DNA and DNA to RNA; both monovalent and divalent cations are required for optimal binding.
Partial sequence of the rat heavy neurofilament polypeptide (NF‐H) Identification of putative phosphorylation sites
Authors: Breen Kieran C. , Robinson Philip A. , Wion Didier , Anderton Brian H.
Published: July 2002

A 3 kb cDNA clone has previously been isolated in this laboratory corresponding to the rat heavy neurofilament polypeptide (NF‐H). This clone, equivalent to approximately 70% of the total mRNA of the protein has been sequenced and shown to contain the carboxy‐terminal region of the message. This contains 51 of the Lys‐Ser‐Pro repeat triplets which are reported to be the site of neurofilament phosphorylation. The sequence obtained was subsequently compared to those of mouse and human NF‐H, showing a homology of approximately 85%. There is, however, one region which is variable between the species, this being the highly phosphorylated region of the protein containing the Lys‐Ser‐Pro triplet repeat.
The hydrolysis of ATP that accompanies actin polymerization is essentially irreversible
Authors: Carlier Marie-France , Pantaloni Dominique , Evans John A. , Lambooy Peter K. , Korn Edward D. , Webb Martin R.
Published: July 2002

The hydrolysis of ATP that accompanies the polymerization of actin occurs on the F‐actin subsequent to the addition of the G‐ATP‐actin subunit to the elongating filament. We now show that this ATP hydrolysis is essentially irreversible. Thus, a large decrease in free energy occurs at the cleavage step, F‐ATP‐actin → F‐ADP‐Pi‐actin.
Resolution of a long lived fluorescence component from D1/D2/ cytochrome b‐559 reaction centres
Authors: Crystall B. , Booth P.J. , Klug D.R. , Barber J. , Porter G.
Published: July 2002

D1/D2/cytochrome b‐559 reaction centres rapidly degrade under continuous illumination. This is a problem in time‐resolved spectroscopy experiments. A long lived fluorescence component assigned to radical pair recombination has been reported previously, but contributing to less than 2% of the fluorescence. This letter reports that sample stability is significantly improved under anaerobic conditions. Fluorescence lifetime measurements described here assign 44% of the total fluorescence yield to a 36.5 ± 2.5 ns lifetime.
Observations on the cytoplasmic and vacuolar orthophosphate pools in leaf tissues using in vivo 31P‐NMR spectroscopy
Authors: Loughman B.C. , Ratcliffe R.G. , Southon T.E.
Published: July 2002

A full understanding of the central role of Pi in photosynthesis requires information on the size of the endogenous Pi pools and the extent to which they interact with each other under different physiological conditions. In vivo 31P‐NMR spectroscopy has the potential to assist in this objective but the early applications of this technique to leaf tissues were disappointing. Here we report 31P‐NMR data from maize and tomato leaf discs that: (i) cast doubt on earlier estimates of the cytoplasmic orthophosphate pool size; and (ii) prove that exogenously supplied D‐mannose can reduce the cytoplasmic Pi level in some circumstances.
Purification of a novel pyruvate kinase from a green alga
Authors: Knowles Vicki L. , Dennis David T. , Plaxton William C.
Published: July 2002

A chloroplast pyruvate kinase has been purified to homogeneity from the green alga Selenastrum minutum. The subunit composition of the enzyme differs markedly from pyruvate kinases from other sources. Subunit and native M
rs were 210 kDa and 235 kDa, respectively, indicating a monomeric structure. Immunological and peptide mapping analyses suggested structural homology to a bacterial, but not higher plant or mammalian pyruvate kinase.
Protective role of CaCl2 against Pb2+ inhibition in photosystem II
Authors: Rashid Abdur , Popovic Radovan
Published: July 2002

The Pb2+ treatment of Photosystem II (PS II) membrane fragments, either intact or depleted in 17 and 23 kDa extrinsic polypeptides, inhibits PS II activity. When CaCl2 was present in the assay, the Pb2+ inhibition was significantly reduced in both types of PS II membranes, suggesting a protective role of CaCl2 against Pb2+ inhibition. However, in either case, the degree of PS II inhibition by Pb2+ was higher in the protein depleted than in intact PS II. It showed that the loss of endogenous Ca2+ induced by polypeptide depletion causes the PS II to be more susceptible to Pb2+. The interaction of Pb2+ with CaCl2 in protein‐depleted PS II was competitive. Our results suggest that Pb2+ competes for binding to the Ca2+ and Cl− active sites in the water‐splitting complex. Since Pb2+ inhibition of PS II activity cannot be reversed by CaCl2 but can be reversed by diphenylcarbazide, we conclude that Pb2+ induced inhibition of PS II activity was mediated via the water‐splitting system.
The monocarboxylate carrier from rat liver mitochondria
Authors: Capuano Ferdinando , Di Paola Marco , Azzi Angelo , Papa Sergio
Published: July 2002

The monocarboxylate (pyruvate) carrier was extracted from rat liver mitochondria with Triton X‐100 in the presence of asolectin and partially purified by chromatography on HTP. The HTP eluate reconstituted in liposomes was shown to catalyze active pyruvatein/acetoacetateout, and acetoacetatein/pyruvateout counter‐exchange. Kinetic characterization of the reconstituted pyruvate carrier was achieved by an original spectrophotometric method consisting of determination of substrate release from proteoliposomes with a coupled enzymatic assay.
Nucleotide sequence of the Yersinia pestis gene encoding F1 antigen and the primary structure of the protein
Authors: Galyov E.E. , Smirnov O.Yu. , Karlishev A.V. , Volkovoy K.I. , Denesyuk A.I. , Nazimov I.V. , Rubtsov K.S. , Abramov V.M. , Dalvadyanz S.M. , Zav'yalov V.P.
Published: July 2002

The plasmid‐located gene cafl encoding the capsular antigen fraction 1 (F1) of Yersinia pestis was cloned and sequenced. The gene codes for a 170 amino acid peptide with a deduced M
r of 17.6 kDa. The signal peptide sequence was highly homologous to the E. coli consensus signal sequence. The F1 was assumed to have β‐sheet structure for the most part. The region located between amino acids 100 and 150 was suggested to contain putative antigenic determinants and to stimulate T cells.
Comparison of the Dl/D2/cytochrome b559 reaction centre complex of photosystem two isolated by two different methods
Authors: Gounaris K. , Chapman D.J. , Booth P. , Crystall B. , Giorgi L.B. , Klug D.R. , Porter G. , Barber J.
Published: July 2002

Photosystem 2 reaction centre complexes prepared either by solubilisation with Triton X‐100 and subsequent exchange into dodecyl maltoside or by a procedure involving a combination of dodecyl maltoside and LiC104, were characterised in terms of chlorophyll a, pheophytin a, β‐carotene and cytochrome b559 content. Time‐resolved chlorophyll fluorescence decay kinetics were measured using both types of complexes. Our data show that the isolated photosystem two reaction centre complex contain, for two pheophytin a molecules, close to six chlorophyll a, two β‐carotene and one cytochrome b559. No major differences were observed in the composition or the kinetic characteristics measured in the samples prepared by the different procedures. Time‐resolved fluorescence measurements indicate that more than 94% of the chlorophyll a in both preparations is coupled to the reaction centre complex.
Protein serine/threonine phosphatases; an expanding family
Authors: Cohen Patricia T.W. , Brewis Neil D. , Hughes Valerie , Mann David J.
Published: July 2002

Five protein serine/threonine phosphatases (PP) have been identified by cloning cDNA from mammalian and Drosophila libraries. These novel enzymes, which have not yet been detected by the techniques of protein chemistry and enzymology, are termed PPV, PP2Bw, PPX, PPY and PPZ. The complete amino acid sequences of PPX, PPY and PPZ and an almost complete sequence of PPV are presented. In the catalytic domain PPV and PPX are more similar to PP2A (57–69% identity) than PPI (45–49% identity), while PPY and PPZ are more similar to PP1 (66–68% identity) than PP2A (44% identity). The cDNA for PP2BW encodes a novel Ca2+/calmodulin‐dependent protein phosphatase only 62% identical to PP2B in the catalytic domain. Approaches for determining the cellular functions of these protein phosphatases are discussed.
Purification and partial characterization of glyoxalase I from a higher plant Brassica juncea
Authors: Deswal Renu , Sopory Sudhir K.
Published: July 2002

The glyoxalase I was purified from Brassica juncea by affinity chromatography on S‐hexyl GSH sepharose 4B, Homogeneity of the protein was confirmed electrophoretically by a silver stained gel. Activity staining on a native starch gel also showed a single band. The effect of glutathione, methylglyoxal, and pH on enzyme kinetics was studied. Magnesium was found to stimulate the enzyme activity.
Thermoregulatory, carboxyatractylate‐sensitive uncoupling in heart and skeletal muscle mitochondria of the ground squirrel correlates with the level of free fatty acids
Authors: Brustovetsky N.N. , Egorova M.V. , Gnutov D.Yu. , Gogvadze V.G. , Mokhova E.N. , Skulachev V.P.
Published: July 2002

Thermoregulatory uncoupling of oxidative phosphorylation has been studied in heart and skeletal muscle mitochondria of ground squirrels. The respiratory rate of mitochondria in the presence of oligomycin was found to be much higher in winter (in hibernating, arousing, or aroused animals) than in summer. This additional respiration is strongly (arousing animals) or completely (hibernating and aroused animals) inhibited by carboxy‐atractylate (CAtr) and bovine serum albumin (BSA). The CAtr‐ and BSA‐induced decreases in the rate of respiration are accompanied by membrane potential increases. The rate of the CAtr‐ and BSA‐sensitive respiration is proportional to the content of free fatty acids which, in the heart, decreases in the order: arousing > aroused = hibernating > summer animals. Maximal respiratory rates observed in the presence of dinitrophenol (arousing > aroused > summer > hibernating animals) do not parallel the fatty acid level. It is assumed that some heat production in the winter animals is due to fatty acid‐induced, ATPIADP‐antiporter‐mediated uncoupling in heart and skeletal muscle mitochondria. The peak of heat production during arousal after hibernation also includes some other stimulatory effect on mitochondrial respiration.
Deletion of internal twenty‐one amino acid residues of Escherichia coli prolipoprotein does not affect the formation of the murein‐bound lipoprotein
Authors: Zhang Wei-Yang , Dai Ren-Ming , Wu Henry C.
Published: July 2002

Mutation pgsA affecting the phosphatidylglycerol phosphate synthesis is lethal for all but certain E. coli strains such as strains deleted for the lpp gene or strains containing unmodifiable prolipoprotein like lppD14. Strain SD312 pgsA3 is tolerant to pgsA mutation, which suggests the lpp alleles in strain SD312 pgsA3 and its parental strain SD12 may be defective. DNA sequence analysis of the lpp genes in Escherichia coli strains SD12 and SD312 pgsA using asymmetric polymerase chain reaction showed that the lpp alleles in these two strains contained a 63 base pair deletion corresponding to the 37th to 57th codons of the wild‐type lpp gene. [3H]Palmitate labeling of strains SD12 and SDS312 showed that the mutant lipoprotein in SD12 strain was modified with lipid, while the prolipoprotein in SD312 was not modified. The shortened mature lipoprotein in SD12 and the lipid‐modified prolipoprotein in globomycin‐treated SD12 were found to be covalently attached to the peptidoglycan, while the unmodified prolipoprotein in SD312 did not form significant amounts of murein‐bound lipoprotein.
Molecular cloning and permanent expression in a neuroblastoma cell line of a fast inactivating potassium channel from bovine adrenal medulla
Authors: García-Guzmán Miguel , Calvo Soledad , Ceña Valentín , Criado Manuel
Published: July 2002

Using a cDNA library from bovine adrenal medulla, we have isolated cDNAs coding for a potassium channel. Then cDNAs encode a 660‐amino acid protein that has a molecular weight of 73.288 kDa and no amino‐terminal signal peptide. We have called it BAK4. Analysis of its sequence reveals close similarity (94% homology) with a recently described potassium channel from rat brain (RCK4) and heart (RHK1). Neuroblastoma cells (Neuro‐2a cell line) were stably transfected with BAK4 DNA. Expression of the DNA was under the control of a heat‐shock promoter. Several clones, that were isolated by neomycin resistance selection, had integrated the plasmid DNA in a stable form. Upon heat induction, these cells produced BAK4 RNA and a potassium outward current, not present in control non‐transfected cells. The current, which was transient and decayed markedly during the duration of 200 ms‐pulses, can be described as ??? potassium current. The expression of these types of channels in brain (RCK4,RHK1), heart (RHK1) and adrenal medulla (BAK4) suggest their possible implication in important functions for the cell.
A novel ubiquinone reductase activity in rat cytosol
Authors: Takahashi Takayuki , Shitashige Miki , Okamoto Tadashi , Kishi Takeo , Goshima Kiyota
Published: July 2002

Ubiquinone (UQ) reductase activity which reduces UQ to ubiquinol (UQH2) in rat tissues was roughly proportional to the UQH2/total UQ ratio in respective tissues. The honest activity was found in the liver, showing the highest UQH2/total UQ ratio. A greater part of liver UQ reductase activity was located in the cytosol. Within a week, the liver UQ reductase activity decreased by 80% even at −20°C. The DT‐diaphorase activity was stable. UQ reductase required NADPH as the hydrogen donor and was not inhibited by a less than 1μM concentration of dicoumarol. There was no stimulation of UQ reductase in the presence of bovine serum albumin nor in Triton X‐100. Yet, both stimulated DT‐diaphorase. As a result, UQ reductase appeared to be a novel NADPH‐UQ oxidoreductase and responsible for the UQ redox state in liver.
Isolation of a novel substrate‐competitive tyrosine kinase inhibitor, desmal, from the plant Desmos chinensis
Authors: Kakeya Hideaki , Imoto Masaya , Tabata Yuji , Iwami Junko , Matsumoto Haruhiko , Nakamura Kazuo , Koyano Takashi , Tadano Kin-ichi , Umezawa Kazuo
Published: July 2002

In the course of a screening program for tyrosine kinase inhibitors, the chloroform extract of a tropical plant, Desmos chinensis, strongly inhibited the enzyme activity. The active substance was purified by silica gel, gel filtration, and finally crystallized. The structure was elucidated by mass spectrometry and X‐ray crystallography to be 8‐formyl‐2,5,7‐trihydroxy‐6‐methylflavanone, and we named it desmal. Desmal competed with peptide substrate and non‐competed with ATP. It inhibited tyrosine kinase in situ in epidermal growth factor (EGF) receptor‐overexpressing NIH3T3 (ER 12) cells. It also inhibited EGF‐induced inositol phosphate formation and morphological changes.
Generation of βA4 from the amyloid protein precursor and fragments thereof
Authors: Dyrks Thomas , Dyrks Elke , Mönning Ulla , Urmoneit Britta , Turner Jonathan , Beyreuther Konrad
Published: July 2002

The cellular mechanisms underlying the generation of βA4 in Alzheimer's disease and its relationship to the normal metabolism of the amyloid protein precursor (APP) are unknown. In this report, we show that expression of the C‐terminal 100 residues of APP, with (SPA4CT) or without (A4CT) a signal sequence in the N‐terminal position, in human neuroblastoma cells results in secretion of a 4 kDa βA4‐like peptide. In A4CT and SPA4CT expressing SY5Y cells, βA4 generation could not be inhibited by the lysosomotropic amines chloroquine and ammonium chloride but was inhibited by brefeldin A, monensin and methylamine. The last also selectively inhibits APP secretion in neuroblastoma cells [1]. The finding that chloroquine and ammonium chloride inhibit βA4 generation from full length APP but not from A4CT and SPA4CT are consistent with the assumption that the two cleavages necessary to generate βA4 operate in two different compartments. Our data suggest the cleavage which generates the C‐terminus of βA4 takes place in the same compartment (late Golgi or endosomal vesicles) in which the APP‐secretase operates.
ATP/ADP antiporter is involved in uncoupling of plant mitochondria induced by low concentrations of palmitate
Authors: Vianello A. , Petrussa E. , Macrì F.
Published: July 2002

Carboxyatractyloside partially restored the transmembrane electrical potential difference (ΔΨ) dissipated by low concentrations of palmitate in pea stem mitochondria. This effect was more marked when mitochondria from sunflower were assayed. It is suggested that the ATP/ADP translocator is involved in the free fatty acid‐induced uncoupling of oxidative phosphorylation in plant mitochondria, only when its level is sufficiently high and the concentration of the fatty acid is low to collapse only partially ΔΨ.
Cloning and expression of a novel form of leukotriene B4 ω‐hydroxylase from human liver
Authors: Kikuta Yasushi , Kusunose Emi , Kondo Tetsuaki , Yamamoto Satoru , Kinoshita Hiroaki , Kusunose Masamichi
Published: July 2002

We have isolated and sequenced a cDNA for human liver LTB4 ω‐hydroxylase. The cDNA encoded a protein of 520 amino acids with a molecular weight of 59,853 Da. The cDNA‐deduced amino acid sequence showed 87.3% homology to that of human polymorphonuelear leukocytes (PMN) LTB4 ω‐hydroxylase (CYP4F3). Northern blot analysis revealed that the mRNA hybridized to the specific cDNA fragment is expressed in human liver, but not in human PMN. The microsomes from yeast cells transfected with the cDNA catalysed the ω‐hydroxylation of LTB4 with a K
m of 44.8 μM. These results clearly show that a new form of the CYP4F LTB4 ω‐hydroxylase exists in human liver.
Regulation of alternative oxidase kinetics by pyruvate and intermolecular disulfide bond redox status in soybean seedling mitochondria
Authors: Umbach Ann L. , Wiskich Joseph T. , Siedow James N.
Published: July 2002

Two factors known to regulate plant mitochondrial cyanide‐resistant alternative oxidase activity, pyruvate and the redox status of the enzyme's intermolecular disulfide bond, were shown to differently affect activity in isolated soybean seedling mitochondria. Pyruvate stimulated alternative oxidase activity at low levels of reduced ubiquinone, shifting the threshold level of ubiquinone reduction for enzyme activity to a lower value. The disulfide bond redox status determined the maximum enzyme activity obtainable in the presence of pyruvate, with the highest rates occurring when the bond was reduced. With variations in cellular pyruvate levels and in the proportion of reduced alternative oxidase protein, a wide range of enzyme activity is possible in vivo.
Amino acid preferences at protein binding sites
Authors: Villar Hugo O. , Kauvar Lawrence M.
Published: July 2002

An analysis of the amino acid distribution at protein binding sites was carried out using 50 diverse macromolecules for which crystallographic data with a bound ligand are available. The purpose of this study is to determine whether differential trends in amino acid distributions exist at binding sites compared to other regions in the proteins. The results indicate that some residues, particularly Arg, His, Trp and Tyr are substantially more frequent at the binding sites, compared to the number of times these residues are present in proteins generally. These effects go beyond the differences seen comparing surface exposed residues to bulk protein. The resemblance in the residue utilization at the binding sites of unrelated proteins restricts the possible types of interactions with ligands, possibly accounting for the repetition of substructural motifs in chemicals with diverse pharmacological action. Further, the use of these diagnostic features may permit identification of ligand binding pockets in a protein structure deduced from sequence information or from data in the absence of a ligand. Some of these findings complement and extend previously described trends for antibody binding sites.
Sequencing of eleven introns in genomic DNA encoding rat glucagon receptor and multiple alternative splicing of its mRNA
Authors: Maget Barbara , Tastenoy Michèle , Svoboda Michal
Published: July 2002

We used the PCR (polymerase chain reaction) to amplify fragments of glucagon receptor DNA from genomic DNA. Sequencing of the subcloned fragments demonstrated that genomic DNA encoding the glucagon receptor spans over 12 exons interrupted by 11 introns. The introns were located mainly at the 5′ end and in the core domain of the glucagon receptor CDS totalling 23 kb. Intron positions were similar to the positions of introns in growth hormone‐releasing hormone receptor and parathyroid hormone receptor, two receptors belonging to the same receptor family as the glucagon receptor. This high number of introns might be the cause of the mRNA polymorphism observed at the 5′ end: when PCR was performed on cDNA using primers amplifying the central or 3′ end cDNA fragments, a single band corresponding to the cloned cDNA was observed. In contrast, if primers amplifying cDNA fragments corresponding to nucleotides −8 to 680 of CDS were used, cDNA fragments of approximately 500 bp, 600 bp, 700 bp, 800 bp and 900 bp were specifically and reproducibly amplified. Sequencing of these fragments showed either incomplete intron removal or splicing at alternative positions. Two of these sequenced variants were translatable in putative glucagon receptor variants: (1) unsplicing of intron III (81 bp) gave an additional 27 amino acid sequence after Lys91 in the N‐terminal domain of the receptor. In the liver, where the normal CDS represented about one third of the mRNA molecules, this mRNA variant represented 18% of total mRNA forms; (2) a 21 bp deletion in exon V giving rise to a putative deletion of 7 amino acids in glucagon receptor (Δ64–84 CDS) was also relatively abundant in the liver (10%). The observed polymorphism of the glucagon receptor mRNA may contribute to the regulation of glucagon receptor expression and perhaps to the heterogeneity of these receptors.
Lack of correspondence between the room‐temperature phosphorescence decay‐components and Trp residues in a series of Trp→Cys or Trp→Phemutants of human carbonic anhydrase II
Authors: Bergenhem Nils C.H. , Schlyer Bruce D. , Steel Duncan G. , Gafni Ari , Carlsson Uno , Jonsson Bengt-Harald
Published: July 2002

The room temperature phosphorescence of native human carbonic anhydrase (CA), and several mutants of this enzyme has been investigated. In these mutants the seven tryptophan residues in the native protein have sequentially been replaced by cysteine or phenylalanine. All of the mutants as well as native CA show room‐temperature tryptophan phosphorescence (RTP) spectra. Surprisingly, only small differences in RTP life‐times are noticeable among these mutants, indicating that there is more than one tryptophan residue with similar phosphorescence decay kinetics in the protein. The present results illustrate the danger in attributing the room temperature phosphorescence of a multi‐tryptophan protein to a particular residue based solely on an analysis of the protein structure.
Poly (ADP‐ribose) polymerase inhibits DNA replication by human replicative DNA polymerase α, δ and ε in vitro
Authors: Eki Toshihiko
Published: July 2002

The influence of poly (ADP‐ribose) polymerase (PARP) and poly ADP‐ribosylation on DNA synthesis supported by human replicative DNA polymerase (DNA pol) α, δ, and ε has been examined using the replication system containing poly(dA)4500‐oligo(dT)12–18 as the template primer. PARP alone inhibited the pol activities in a dose‐dependent manner even in the presence of the accessory factors for DNA pol δ, proliferating cell nuclear antigen (PCNA) and activator 1 (A1; RF‐C). Both DNA pol α and ε activities were decreased approximately 10‐fold under the poly ADP‐ribosylating condition. In contrast, DNA synthesis by DNA pol δ holoenzyme was not affected by poly ADP‐ribosylation like prokaryotic DNA pol's. The analysis of poly(dT) formed by DNA pol α and ε indicated that poly ADP‐ribosylation mainly reduced the frequency of replication. These observations suggest a possibility that PARP acts as a negative regulator for the initiation of DNA replication upon cellular DNA damage.
Effects of adenylyl cyclase‐linked neuropeptides on the expression of ciliary neurotrophic factor‐mRNA in cultured astrocytes
Authors: Nagao Hiroshi , Matsuoka Ichiro , Kurihara Kenzo
Published: July 2002

Ciliary neurotrophic factor (CNTF) is a molecule which has profound effects on various neural cell types. In the central nervous system, expression of CNTF‐mRNA is highly concentrated in olfactory bulb. In the present study, we examined the regulatory mechanism of CNTF‐mRNA expression in cultured astrocytes from newborn rat brain. Cultured astrocytes from new born rat brain expressed CNTF‐mRNA at levels comparable to the level in olfactory bulb in vivo. Treatment of the astrocytes with forskolin, an activator of adenylyl cyclase, led to a decrease of CNTF‐mRNA level. The effect of forskolin was mimicked by cAMP‐linked agonists, such as VIP, PACAP, isoproterenol and dopamine. Cycloheximide, an inhibitor of protein synthesis, did not abolish the forskolin‐induced decrease of CNTF‐mRNA. Measurement of the half‐life of CNTF‐mRNA in the presence of actinomycin D, an inhibitor of transcription, indicated that the degradation of CNTF‐mRNA is not destabilized by the forskolin‐treatment. These data taken together suggest that the cAMP‐induced suppression of CNTF‐mRNA is mainly caused by the inhibition of CNTF gene transcription.
Human CuZn superoxide dismutase enzymatic activity in cells is regulated by the length of the mRNA
Authors: Kilk Ann , Laan Maris , Torp Aivar
Published: July 2002

Single functional human CuZnSOD gene encodes two species of mRNA differing in size by 200 nucleotides in the 3′‐untranslated region (UTR). We studied the expression of the CuZnSOD cDNA with different 3′‐ and 5′‐UTR. Deletion in the 5′‐end does not affect the expression of the enzyme, however, deletion in the 3′‐UTR decreases the level of expression of CuZnSOD. The plasmids containing the long CuZnSOD cDNA with all polyadenylation signal sequences utilize primarily the last polyadenylation site and give a long mRNA, which produces three times more enzyme than the short mRNA lacking the last polyadenylation site and the AU‐rich region.
Immunocytochemical study of phenotypic plasticity of cultured dorsal root ganglion neurons during development
Authors: Chauvet N. , Drian M.‐J. , Privat A.
Published: July 2002

Rat dorsal root ganglia (DRG) were cultured from different stages of development ranging from embryonic day‐14 to adult. The expression of eight neurotransmitter phenotypes was examined with immunocytochemical detection and the percentages of each phenotype were calculated with reference to the whole neuronal population defined by the expression of neuron‐specific enolase (NSE). The expression of peptides, calcitonin gene‐related peptide (CGRP), substance P (SP), cholecystokinin (CCK) and neuropeptide Y (NPY) was always present whatever the age at onset of the cultures. Although the percentage of CGRP remained stable, that of the other peptides declined progressively. Their in‐vitro expression did not differ marked from that found in vivo. Another group of neurotransmiiters, including 5‐hydroxytryptamine (5‐HT), thyrotropin‐releasing hormone (TRH) and gamma‐aminobutyric acid (GABA) was never expressed in situ in DRG neurons. In culture, they were expressed in a high percentage of neurons, especially for 5‐HT and TRH, and they showed a similar evolution, with a decrease at early postnatal ages followed by a further increase. This profile suggests that the expression of these transmitters is strongly environment‐dependent and may be repressed in situ. Finally, somatostatin (SOM) was found only in cultures prepared from adult tissues, whereas it was present in situ from the embryo onwards. The expression of this peptide would thus require a stabilization by a long exposure to environmental factors.

We can conclude that the great diversity of phenotypic expression found in DRG neurons in situ is the result of a wide variety of influences occurring at different stages of development in a large potential repertory present in these neurons.
Atypical neuroleptics: their contribution to the rehabilitation of schizophrenic patients
Authors: Vanelle JM , Brochier T , Baylé FJ , Lôo H
Published: July 2002

Therapeutic effects of neuroleptics (NLP) in schizophrenia are well established in numerous international studies. Varying modalities of evolution under NLP treatment are described and require a clinical analysis. Neuroleptics are a necessary but insufficient condition for the therapy of schizophrenia. Via NLP treatment, other therapeutic approaches, such as psychotherapy, or sociotherapy become more effective. Consecutively, we present the effects of two compounds with dopaminergic selective activity (amisulpride, remoxipride) and of two other compounds with non-selective activity (risperidone, clozapine) regarding rehabilitation in schizophrenic patients. Data were collected from international studies with a medico-economic perspective and from French experience of clozapine in refractory schizophrenic patients. All these data stress the importance of several areas of study: clinical features, everyday life consequences and therapeutical effects observed with the atypical neuroleptics.