bluuebunny/crossref_metadata_2025 huggingface dataset:
Adaptive neural PD controllers for mobile manipulator trajectory tracking
Authors: Hernandez-Barragan Jesus , D. Rios Jorge , Gomez-Avila Javier , Arana-Daniel Nancy , Lopez-Franco Carlos , Alanis Alma Y.
Published: February 2021

Artificial intelligence techniques have been used in the industry to control complex systems; among these proposals, adaptive Proportional, Integrative, Derivative (PID) controllers are intelligent versions of the most used controller in the industry. This work presents an adaptive neuron PD controller and a multilayer neural PD controller for position tracking of a mobile manipulator. Both controllers are trained by an extended Kalman filter (EKF) algorithm. Neural networks trained with the EKF algorithm show faster learning speeds and convergence times than the training based on backpropagation. The integrative term in PID controllers eliminates the steady-state error, but it provokes oscillations and overshoot. Moreover, the cumulative error in the integral action may produce windup effects such as high settling time, poor performance, and instability. The proposed neural PD controllers adjust their gains dynamically, which eliminates the steady-state error. Then, the integrative term is not required, and oscillations and overshot are highly reduced. Removing the integral part also eliminates the need for anti-windup methodologies to deal with the windup effects. Mobile manipulators are popular due to their mobile capability combined with a dexterous manipulation capability, which gives them the potential for many industrial applications. Applicability of the proposed adaptive neural controllers is presented by simulating experimental results on a KUKA Youbot mobile manipulator, presenting different tests and comparisons with the conventional PID controller and an existing adaptive neuron PID controller.
Regucalcin expression profiles in veal calf testis: validation of histological and molecular tests to detect sex steroids illicit administration
Authors: Benedetto Alessandro , Biasibetti Elena , Beltramo Chiara , Audino Valentina , Peletto Simone , Bozzetta Elena Maria , Pezzolato Marzia
Published: February 2021

Background

Sex steroids administration in meat producing animals is forbidden within the EU to preserve consumers’ safety, but continuous monitoring to identify resurgence of their misuse is needed. Among biomarkers related to sex steroids abuse in veal calves the regucalcin (RGN) mRNA perturbations in testis have been described in RNAlater samples. To setup novel diagnostic method, to update current tests available in National Residue Control Plans (NRCPs) and in legal dispute when illicit practices on farm animals are suspected, the reliability of RGN profiling was assessed by histological and molecular techniques.

Methods

Formalin fixed paraffin embedded (FFPE) testis samples, chosen being the most effective preservation strategy adopted by histological NRCPs and allowing easier retrospective analysis if required by legal disputes, were analyzed from veal calves treated with nandrolone, 17β-estradiol and a cocktail of the two hormones. RGN levels were determined by quantitative Real Time PCR and Immunohistochemistry assays. Test performances were assessed and compared by multiple ROC curves.

Results

Both tests resulted sensitive and specific, allowing to enrich, in future field investigation, novel integrated diagnostic protocols needed to unveil sex steroid abuse.

Discussion

Developed RT-qPCR and IHC methods confirmed RGN as a useful and robust biomarker to detect illegal administration of sex steroid hormones in veal calves. The developed methods, successfully applied to ten years old FFPE blocks, could allow both retrospective analysis, when supplementary investigations are requested by authorities, and future implementation of current NRCPs.
Stability-indicating UHPLC and TLC-densitometric Methods for the Determination of Tiamulin Fumarate
Authors: Aziz Shimaa E. Abdel , Ahmed Nermin S. , Razeq Sawsan A. Abdel
Published: February 2021

Two novel stability-indicating methods were developed for the determination of tiamulin fumarate. in the presence of its degradation products. The first was UHPLC-UV method. Efficient separation was achieved by isocratic elution with a mobile phase of 0.1% aqueous ortho-phosphoric acid (pH 3.5 ± 0.5) and methanol (20:80, v/v) with UV detection at 210 nm. Linearity was obtained in the range of 0.5-10.0 µg mL-1 with mean accuracy of 100.40 ± 0.71. The second method was a TLC-densitometric evaluation of a thin-layer chromatogram of the intact drug using a mobile phase of methanol: pentanol: ethyl acetate: 16.5% ammonia (5:4:2:4, by volume). The TLC-plates were scanned densitometrically at 220 nm where Rf values were 0.58, 0.48 and 0.74 for tiamulin F, its acidic and oxidative degradants, respectively. Moreover, the plates were sprayed with 16% sulfuric acid, heated at 105°C for 10 min. where a yellow-coloured band appeared corresponding to the intact drug was scanned densitometrically at 450 nm. While the bands of the two degradants were no longer observed anymore. Tiamulin F was determined in the range of 1.0-10.0 μg/band with mean accuracy of 100.27% ± 1.47 at 220 nm and 99.93% ± 1.38 at 450 nm. The proposed methods were successfully applied for the determination of drug in marketed oral solution. The obtained results were statistically analyzed and found to be in accordance with those obtained by a reported method.
Mobilization of maternal mRNA in amphibian eggs with special reference to the possible role of membraneous supramolecular structures
Authors: Shiokawa Koichiro
Published: July 2002

Current knowledge of the mechanism of mobilization of maternal mRNA is summarized herein and a working hypothesis has been constructed to explain the mechanism on the assumption that the mRNA enters the cytoplasm in association with the cytoplasmic membraneous structures and is then stored in the structures until liberation and relocation at the step of oocyte maturation. An extensive turnover of poly(A) sequences as well as the occurrence of repetitive sequences in the maternal mRNA may be relevant to mRNA activation.
The action of 1,4‐β‐D‐glucan cellobiohydrolase on Valonia cellulose microcrystals
Authors: Chanzy Henri , Henrissat Bernard , Vuong Roger , Schülein Martin
Published: July 2002

The interaction between 1,4‐β‐D‐glucan‐cellobiohydrolase I (CBHI) from Trichoderma reesei and microcrystalline cellulose from Vallonia macrophysa was investigated by electron microscopy which allows to visualize the individual enzymes and their strong adsorption at the substrate surface. The microcrystals are slowly degraded and decrystallized by erosion and fibrillation while soluble products are released and characterized by HPLC. CBHI is thus able to break down Valonia microcrystals without the help of any endo‐β‐1,4‐glucanase activity.
Effects of mutations and of ionophore on chlororespiration in Chlamydomonas reinhardtii
Authors: Bennoun Pierre
Published: July 2002

It is shown that electron‐transport elements of the photosynthetic chain located between plastoquinone and photosystem I centers are not involved in oxidation of plastoquinone in the chlororespiratory pathway. The ionophore dicyclohexyl‐18‐crown‐6 is shown to induce a reduction of the plastoquinone pool in the dark. This is interpreted as indicating the existence of a coupling site in the chlororespiratory pathway between NAD(P)H and plastoquinone.
Sarcolemmal phospholamban is phosphorylated in isolated rat hearts perfused with isoprenaline
Authors: Huggins John P. , England Paul J.
Published: July 2002

Phosphorylation of phospholamban in cardiac sarcolemma is implicated in the increased influx of Ca2+ through the slow calcium channel induced by catecholamines. A method is described for the preparation of highly purified sarcolemmal vesicles from rat heart, and this has been used to examine the phosphorylation of phospholamban in 32Pi‐perfused rat hearts. Phospholamban phosphorylation is increased 3‐fold after 30 s of perfusion with 0.1 μM isoprenaline. The time course of this increase precedes the inotropic response by 5–10 s.
Peptide uptake in germinating barley embryos involves a dithiol‐dependent transport protein
Authors: Walker Smith David J. , Payne John W.
Published: July 2002

An active transport system for small peptides occurs in the scutellar membrane of germinating barley and serves to move the products of partial hydrolysis of storage proteins from the endosperm into the growing embryo. Transport of peptides, but not amino acids or glucose, is inhibited by the thiol reagents, N‐ethylmaleimide and p‐chloromercuribenzene sulphonic acid (PCMBS). Peptide substrates protect against PCMBS inactivation. The dithiol‐specific reagent, phenylarsine oxide (PAO) also inhibits. The reducing agent, dithiothreitol, reverses the inactivation caused by PCMBS and PAO. We conclude that the peptide transport system contains a redox‐sensitive, dithiol‐dependent protein.
Axonal transport of muscarinic receptors in vesicles containing noradrenaline and dopamine‐β‐hydroxylase
Authors: Laduron Pierre M.
Published: July 2002

Presynaptic muscarinic receptors labeled with [3H]dexetimide and noradrenaline in dog splenic nerves accumulated proximally to a ligature at the same rate of axonal transport. After fractionation by differential centrifugation, specific [3H]quinuclidinyl benzilate or [3H]dexetimide binding revealed a distribution profile similar to that of dopamine‐β‐hydroxylase and noradrenaline. Subfractionation by density gradient centrifugation showed two peaks of muscarinic receptors; the peak of density 1.17 contained noradrenaline and dopamine‐β‐hydroxylase whereas that of density 1.14 was devoid of noradrenaline. Therefore the foregoing experiments provide evidence that presynaptic muscarinic receptors are transported in sympathetic nerves in synaptic vesicles which are similar to those containing noradrenaline and dopamine‐β‐hydroxylase. This suggests a possible coexistence of receptor and neurotransmitter in the same vesicle.
Enkephalin degrading enzymes are present in the electric organ of Torpedo californica
Authors: Altstein Miriam , Dudai Yadin , Vogel Zvi
Published: July 2002

Two proteolytic activities that degrade [Leu5]enkephalin were found in Torpedo californica electric organ. One is a soluble aminopeptidase that degrades enkephalin at the Tyr1—Gly2 peptide bond, and the second is an endopeptidase that degrades enkephalin at the Gly3—Phe4 peptide bond. The aminopeptidase is inhibited by low concentrations of puromycin and bestatin. More than 60% of the endopeptidase is associated with the particulate fraction and is almost completely inhibited by low concentrations of captopril (SQ 14225) or SQ 20881 (potent inhibitors of angiotensin converting enzyme). Thiorphan and phoshoramidon (potent enkephalinase inhibitors) are much less effective. The pattern of cleavage and inhibition of the particulate endopeptidase thus resembles that of angiotensin converting enzyme.
A model for the secondary structure of β‐lactamases
Authors: Bunster Marta , Cid Hilda
Published: July 2002

A 3‐dimensional model, common for the secondary structures of four β‐lactamases obtained from Escherichia coli, Bacillus licheniformis, Bacillus cereus and Staphylococcus aureus, is proposed. The predictions of the structures were made by the hydrophobicity profiles method complemented by the modified Chou and Fasman's method. The model proposed presents 56% constancy and can be described as a 2‐domain structure, in agreement with low resolution X‐ray data reported for the E. coli enzyme. The model would explain how a common function can be performed by enzymes of very different sizes, composition and sequence.
An unusual lectin from stinging nettle (Urtica dioica) rhizomes
Authors: Peumans Willy J. , De Ley Marc , Broekaert Willem F.
Published: July 2002

An unusual lectin has been isolated from stinging nettle (Urtica dioica L.) rhizomes. It is a small (8.5 kDa) monomeric protein with high contents of glycine, cysteine and tryptophan. The U. dioica agglutinin (UDA) is not blood group‐specific and is specifically inhibited by N‐acetylglucosamine oligomers. As compared to other plant lectins, UDA has a very low specific agglutination activity. Nevertheless, it induces HulFN‐γ in human lymphocytes at concentrations comparable to those of other inducers.
Carbonic anhydrase located on cell surface increases the affinity for inorganic carbon in photosynthesis of Dunaliella tertiolecta
Authors: Aizawa Katsunori , Miyachi Shigetoh
Published: July 2002

Low‐CO2 cells of Dunaliella tertiolecta had higher carbonic anhydrase (CA) activity on their surfaces than inside. The treatment with subtilisin completely abolished CA activity on the cell surface, while the activity inside the cells was not affected by this treatment. Correspondingly, the apparent K
m (NaHCO3) for photosynthesis was increased two‐fold, while V
max was not affected by this treatment. The apparent K
m(NaHCO3) was lowered to the original level when CA was added to the suspension of subtilisin‐treated cells. These results indicate that CA on the cell surface enhances the affinity for inorganic carbon in photosynthesis of D. tertiolecta.
CMP‐sialic acid, the sole sialosyi donor, is intra‐axonally transported
Authors: Igarashi Michihiro , Komiya Yoshiaki , Kurokawa Masanori
Published: July 2002

N‐Acetyl‐D‐[6‐3H]mannosamine was injected into the 9th dorsal root ganglion of Xenopus laevis and the intra‐axonal transport of chloroform/methanol‐soluble radioactivity was analyzed using thin‐layer chromatography coupled with fluorography. Three radioactive groups were distinct in consecutive segments of the sciatic nerve. The first is due to N‐acetyl‐D‐mannosamine itself which labels the nerve uniformly, but does not seem to migrate within axons. The second group, representing most probably CMP‐sialic acid, migrates at about 8 at 15°C. The third is a species of ganglioside uniquely present in the frog nerve, and this migrates at 1–3 . Our demonstration of the intra‐axonal transport of CMP‐sialic acid affords direct support to the contention that sialosylation of the ganglioside can occur in axon terminals.
Electrochemical detection of biogenic amines following acylation by N‐hydroxysuccinimide esters
Authors: Jacobson Kenneth A. , Marshall Thomas , Mine Kazanori , Kirk Kenneth L. , Linnoila Markku
Published: July 2002

A general methodology for the selective derivatization of amines, to enable quantitation by high pressure liquid chromatography with electrochemical detection, is presented. N‐Hydroxysuccinimide active esters present in large excess are suitably mild acylating agents to derivatize selectively trace quantities of amines for electrochemical detection. The 2 separate problems of extraction yield and detectability can be solved by this derivatization method. Due to its lipophilicity the resulting N‐acylated amine, as demonstrated with serotonin, is extracted efficiently into organic solvents during sample preparation for chromatography. Moreover, the acyl group introduced can be designed to be electroactive, thus extending the procedure to amines not readily oxidized, e.g., histamine and phenylethylamine.
Increasing intracellular pH of sea urchin sperm with NH4Cl induces Ca2+ uptake and acrosome reaction in the absence of egg jelly
Authors: Garcia-Soto J. , de la Torre L.de , Darszon A.
Published: July 2002

Intracellular alkalinization and Ca2+ uptake accompany the egg jelly‐induced acrosome reaction of sea urchin sperm. Here, sperm were incubated in NF4Cl‐containing seawater maintained at its physiological pH (8.0). Under these conditions, 20 mM NH4C1 induced intracellular alkalinization, Ca2+ uptake and acrosome reaction in the absence of egg jelly. The NH4Cl‐triggered acrosome reaction required external Ca2+. Our results indicate that internal alkalinization activates Ca2+ influx into sperm possibly through a pH sensitive Ca2+ channel. Thus egg jelly could first stimulate the sperm plasma membrane Na+/H+ exchanger inducing intracellular alkalinization which would then activate Ca2+ uptake and acrosome reactions.
Amino acid sequence of an amyloidogenic Bence Jones protein in myeloma‐associated systemic amyloidosis
Authors: Tonoike Hiroshi , Kametani Fuyuki , Hoshi Akihiko , Shinoda Tomotaka , Isobe Takashi
Published: July 2002

The complete amino acid sequence of an amyloidogenic Bence Jones protein(NIG‐84) from an individual with myeloma‐associated systemic amyloidosis has been determined. The protein, with a blocked N‐terminus, represents a complete light chain consisting of 217 residues and it has a structural feature characteristic of the Vλ II subgroup. In addition to a two‐residue insertion at positions 28 and 29, it has an additional rare insertion of alanine at position 100. NIG‐84 is an example of the first complete sequence presented for the amyloidogenic Bence Jones protein of the Vλ II subgroup.
Reversed‐phase ion‐pair Chromatographic separation of ribulose 1,5‐bisphosphate from 3‐phosphoglycerate and its application as a new enzyme assay for RuBP carboxylase/oxygenase
Authors: Jakob Robert , Saenger Wolfram
Published: July 2002

HPLC
Ion‐pair chromatography
Ribulose 1,5‐bisphosphate
Ribulose 5‐monophosphate
Ribulose‐1,5‐bisphosphate carboxylase/oxygenase
3‐Phosphoglycerate
On the ubiquitous presence of histone acetyltransferase B in eukaryotes
Authors: Salvador M.Luisa , Sendra Ramón , López-Rodas Gerardo , Tordera Vicente , Franco Luis
Published: July 2002

Histone acetyltransferase B activity has been found in pea (Pisun sativum) seedlings. The enzyme has been partially purified and it has been found that it is highly specific for H4. The results confirm that histone acetyltransferase B occurs in 3 eukaryotic kingdoms.
Amino acid sequence of human liver cathepsin B
Authors: Ritonja Anka , Popovic Tatjana , Turk Vito , Wiedenmann Karin , Machleidt Werner
Published: July 2002

The complete amino acid sequence of cathepsin B (EC 3.4.22.1) from human liver was determined. The 252‐residue sequence was obtained by automated solid‐phase Edman degradation of the light and heavy chain resulting from limited proteolysis of the single‐chain enzyme and of fragments produced by cyanogen bromide and enzymatic cleavage of the heavy chain. Human liver cathepsin B has 83.7% identical residues with the corresponding enzyme from rat liver. Comparison of both mammalian cathepsin B sequences with the sequence of papain provides further evidence that lysosomal and plant cysteine proteinases have evolved from a common ancestor and share a similar catalytic mechanism.
Degradation of α‐actinin during Ca2+‐sensitive proteolysis of myofibrils
Authors: Kulesza-Lipka Dorota , Jakubiec-Puka Anna
Published: July 2002

The noted loss of α‐actinin from the Z‐line of myofibrils during post‐mortem autolysis, probably following the action of calcium‐activated protease, has previously been attributed to its release without degradation. This report shows that in isolated myofibrils α‐actinin is proteolysed in a Ca2+‐sensitive manner presumably via the action of calcium‐activated protease.
Is the C‐terminal flanking peptide of rat cholecystokinin double sulphated?
Authors: Adrian T.E. , Domin J. , Bacarese-Hamilton A.J. , Bloom S.R.
Published: July 2002

A specific radioimmunoassay was developed to the predicted nine amino acid C‐terminal flanking peptide of cholecystokinin (peptide serine serine, PSS). In aqueous extracts of rat brain, PSS was undetectable unless the extracts were first treated with arylsulphatase, which also resulted in desulphation of cholecystokinin. The reverse‐phase HPLC analysis of partially desulphated extracts showed the presence of two peaks intermediate to the naturally occurring and the completely desulphated forms. It is therefore proposed that the CCK‐flanking peptide PSS has both tyrosine residues sulphated.
Localization of the N‐terminal regions of the B870α,β and of reaction center L polypeptides on the cytoplasmic surface of the chromatophores of Rhodopseudomonas capsulata
Authors: Tadros Monier Habib , Frank Rainer , Drews Gerhart
Published: July 2002

Chromatophores (intracytoplasmic inside‐out membrane vesicles) from the mutant strain Ala+ of Rhodopseudomonas
capsulata were treated with proteinase K in order to determine the orientation of the pigment‐binding polypeptides of the photochemical reaction center and of the light‐harvesting complex B870. Nine amino acid residues of the B870α polypeptide were cleaved up to position Leu9‐Val10 of the N‐terminus and 22 residues up to position Val22‐Tyr23 of the β‐chain N‐terminus. From the reaction center L‐chain the N‐terminus up to position 27 (Val) was split off. The C‐termini of the B870α and β polypeptides including the hydrophobic α‐helical transmembrane portion remained intact. It is concluded that the N‐terminal region of the pigment‐binding polypeptides α and β of the B870 light‐harvesting complex and of the L‐chain of the reaction center point to the cytoplasm while the C‐termini of the B870α,β polypeptides are exposed or pointing toward the periplasmic membrane surface.
Selective detection of rapid motions in spectrin by NMR
Authors: Fung L.W.-M. , Lu H.-Z. , Hjelm R.P. , Johnson M.E.
Published: July 2002

Human erythrocyte spectrin molecules exhibit relatively sharp (30–50 Hz) proton NMR signals in the aliphatic region. A standard solvent presaturation pulse sequence that also partially suppresses the broad envelope from protons with rigid structures in spectrin and selectively enhances the sharp resonances has been used to characterize the behavior of these resonances. The overall resonance pattern strongly resembles that of the denatured spectrin. The observed spectra are also quite similar to the line‐broadened spectrum from a mixture of amino acids that corresponds to the composition of the spectrin molecule. These data indicate the existence of regions exhibiting rapid internal motions within the intact spectrin molecule, and suggest that the amino acid composition of the residues giving rise to the sharp resonances is quite similar to that of the full spectrin molecule.
Local synthesis of plasminogen by the seminiferous tubules of the testis
Authors: Saksela O. , Vihko K.K.
Published: July 2002

Plasminogen activation occurs through conversion of plasminogen to plasmin by plasminogen activators. In adult mammals, liver has been the only known site for plasminogen synthesis. Seminiferous tubules secrete plasminogen activator, but are behind a barrier that excludes the entrance of many macromolecules to this tissue. Therefore, it became of interest to study the existence of plasminogen in this system. After metabolic labeling, a 90 kDa lysine‐binding polypeptide was found. This protein could be immunoblotted by anti‐plasminogen antibody, and was shown to possess urokinase‐dependent proteolytic activity. The findings suggest that plasminogen is synthesized by seminiferous tubules. We propose that local plasminogen synthesis may occur in tissues which are separated by specific anatomical barriers.
Muscarinic M2‐receptors enhance polyphosphoinositol release in rat gastric mucosal cells
Authors: Pfeiffer Andreas , Paumgartner Gustav , Herz Albert
Published: July 2002

Muscarinic receptor types and their effects on the inositol phosphate second messenger system were studied in enzymatically dispersed rat gastric mucosal cells. Radioreeeptor binding studies indicated the presence of a single class of binding sites (4860 ± 875 ) and affinities of 0.42 ± 0.12 nM, 176 ± 32 nM and 13 μM for N‐methylscopolamine, pirenzepine and carbachol, respectively. In cells prelabelled with myo‐[3H]inositol carbachol induced a dose‐dependent increase in inositol‐1‐phosphate in the presence of lithium with an ED50 of 10 μM which was antagonized by atropine and pirenzepine (IC50 9 and 700 nM, respectively). Carbachol stimulated amino[14C]pyrine uptake, used as a measure of acid secretion, with an ED50 of 10 μM. The good correlation between these responses suggests a role for inositol phosphates in the muscarinic M2‐receptor mediated acid secretion.
Isolation of a cDNA for the rat heavy neurofilament polypeptide (NF‐H)
Authors: Robinson Philip A. , Wion Didier , Anderton Brian H.
Published: July 2002

We have isolated from a rat brain λgtll expression library two overlapping cDNA clones of sizes 2.5 and 3.0 kb corresponding to the heavy neurofilament polypeptide (NF‐H). The 2.5 kb insert apparently represents virtually the whole of the C‐terminal tail, the 3.0 kb insert also encodes the conserved epitope for the monoclonal antibody, anti‐IFA. The identity of the cDNAs was established by comparison of the predicted amino acid sequence with the known partial amino acid sequence of porcine NF‐H. A repeat peptide sequence that may be a multiphosphorylation site was identified in the C‐terminal non‐helical tail.
Ectopic synthesis of high‐Mr calcitonin by the BEN lung carcinoma cell line reflects aberrant proteolytic processing
Authors: Riley John H. , Edbrooke Mark R. , Craig Roger K.
Published: July 2002

Cloning and nucleotide sequence analysis of the human calcitonin mRNA from the BEN lung carcinoma cell line, a cell line known to secrete high‐M
r forms of calcitonin, showed no difference in the Coding region at the nucleotide level compared with calcitonin mRNA isolated from medullary thyroid carcinoma which secretes calcitonin monomer. Therefore, the secretion of high‐M
r forms of calcitonin reflects the absence or limited activity of proteolytic processing enzymes within the secretory pathway of this cell line. In all other respects, as judged by RNA blotting and S1 mapping, calcitonin/α‐CGRP expression was identical to that found in medullary thyroid carcinoma, including the differential use of an alternative splice donor site within intron 1. The BEN cell line also produces low levels of α‐CGRP mRNA and secretes CGRP antigenic determinants. Analysis of plasma CGRP levels in 12 patients with anaplastic lung carcinoma showed elevated levels in 11 of these, suggesting that CGRP may be an important diagnostic marker for this disease.
The acid‐stable proteinase inhibitor of human mucous secretions (HUSI‐I, antileukoprotease)
Authors: Seemüller Ursula , Arnhold Marianne , Fritz Hans , Wiedenmann Karin , Machleidt Werner , Heinzel Regina , Appelhans Heribert , Gassen Hans-Günter , Lottspeich Friedrich
Published: July 2002

The complete amino acid sequence of human antileukoprotease has been determined by direct sequencing of the inhibitory active protein isolated from seminal plasma (HUSI‐I) and by sequence analysis of cDNA reverse‐transcribed from mRNA prepared from cervical tissue. The inhibitor (M
r 11 726) consists of 107 amino acid residues including 16 cysteines presumably forming disulfide bonds. The molecule comprises two consecutive domains which are homologous to each other, to the second domain of the basic protease inhibitor from Red Sea turtle (chelonianin) and to both domains of the whey proteins of rat and mouse. Both domains contain a pattern of cysteines known as the ‘four‐disulfide‐core’ that has also been found in wheat germ agglutinin and neurophysin.
Computer‐aided design and physiological testing of a luteinising hormone‐releasing hormone analogue for ‘adjuvant‐free’ immunocastration
Authors: Morrison Christopher A. , Fishleigh Robert V. , Ward David J. , Robson Barry
Published: July 2002

An analogue of LHRH containing an extension of Gly‐Cys at the carboxyl‐terminus has been designed to permit reproducible coupling to a suitably modified carrier via a thioether bond. Potential energy calculations indicated that this analogue adopted a conformation in solution virtually identical to the type II′ turn around Gly‐6—Leu‐7 predicted for native LHRH. Intradermal administration of a conjugate of this analogue with purified protein derivative of tuberculin to male rats previously primed with BCG vaccine rapidly led to complete testicular regression. This adjuvant‐free immunisation protocol may represent an alternative to castration for the veterinary control of reproductive function.
Phycobilisome‐associated glycoproteins in the cyanobacterium Anacystis nidulans R 2
Authors: Riethman Harold C. , Mawhinney Thomas P. , Sherman Louis A.
Published: July 2002

Four phycobilisome‐associated proteins were found to be specifically reactive with the lectin concanavalin A after subunits of isolated Anacystis nidulans R 2 phycobilisomes were separated on polyacrylamide gels and transferred onto nitrocellulose. The concanavalin A‐reactive phycobilisome components have proposed functions related to the orientation, assembly, and membrane attachment of the phycobilisome. Chemical analysis of the total isolated phycobilisome material indicated the presence of glucose (approx. 1.5% by wt) and N‐acetylgalactosamine (0.15% by wt), consistent with our proposal that the concanavalin A‐reactive polypeptides contain covalently linked, glucose‐containing polysaccharides.
Nicotinic acetylcholine receptors in cultured neurons from the hippocampus and brain stem of the rat characterized by single channel recording
Authors: Aracava Y. , Deshpande S.S. , Swanson K.L. , Rapoport H. , Wonnacott S. , Lunt G. , Albuquerque E.X.
Published: July 2002

Single channel recording techniques have been applied to neurons cultured from the hippocampus and the respiratory area of the brain stem of fetal rats in order to search for nicotinic acetylcholine receptors (nAChR) in the central nervous system. In addition to acetylcholine (ACh), the potent and specific agonist (+)‐anatoxin‐a was also used to characterize nicotinic channels. nAChRs were concentrated on the somal surface near the base of the apical dendrite, and in some patches their density was sufficient to record 2 or more channel openings simultaneously. Although a multiplicity of conductance states was also evident, the predominant population showed a single channel conductance of 20 pS at 10°C. Thus, these neuronal nAChRs resembled the embryonic or denervated‐type nAChRs in muscle. However, channel opening and closing kinetics were faster than reported for similar conductance channels in muscle. Therefore the nicotinic channels described here are similar but not identical to those of the well‐characterized muscle nAChR, in agreement with biochemical, pharmacological, and molecular genetic studies on brain AChR.
The geometries of interacting arginine‐carboxyls in proteins
Authors: Singh J. , Thornton J.M. , Snarey M. , Campbell S.F.
Published: July 2002

The geometries are reported for interacting arginine‐carboxyl pairs obtained from 37 high resolution protein structures solved to a resolution of 2.0 Å or better. The closest interatomic distance between the guanidinium and carboxyl is less than 4.2 Å for 74 arginine and carboxyl groups, with the majority of these lying within hydrogen‐bonding distance (2.6–3.0 Å). Interacting pairs have been transformed into a common orientation, and arginine‐carboxyl, and carboxyl‐arginine geometries have been calculated. This has been defined in terms of the spherical polar angles Tθ, Tϕ, and the angle P, between the guanidinium and carboxyl planes. Results show a clear preference for the guanidinium and carboxyl groups to be approximately coplanar, and for the carboxyl oxygens to hydrogen bond with the guanidinium nitrogens. Single nitrogen‐single oxygen is the most common type of interaction, however twin nitrogen‐twin oxygen interactions also occur frequently. The majority of these occur between the carboxyl oxygens and the NH1 and NE atoms of the arginine. and are only rarely observed for NH1 and NH2. The information presented may be of use in the modelling of arginine‐carboxyl interactions within proteins.
The human preproapolipoprotein C‐II gene
Authors: Fojo Silvia S. , Law Simon W. , Brewer H.Bryan
Published: July 2002

The complete nucleic acid sequence of human preproapolipoprotein (apo) C‐II has been determined from 2 apoC‐II clones isolated from 2 different human genomic DNA libraries. The cloned fragments were approx. 14 and 18 kb long, and sequence analysis established that the apoC‐II gene consists of 3338 nucleotides containing 3 intervening sequences of 2391, 167, and 298 bases. The first intron is located within the 5'‐untranslated region of apoC‐II and contains 4 Alu type sequences. The second intron interrupts the codon specifying amino acid —11 of the apoC‐II signal peptide. The last intron, which contains a 38 bp sequence which is repeated 6 times, interrupts the codon specifying for amino acid +44 of the mature apolipoprotein.
The diversity of mercury reductases among mercury‐resistant bacteria
Authors: Bogdanova E.S. , Mindlin S.Z. , Kalyaeva E.S. , Nikiforov V.G.
Published: July 2002

Two immunologically non‐cross‐reactive types of mercury reductases were found among Gram‐negative and two among Gram‐positive mercury‐resistant environmental bacteria. Mercury reductases were further discriminated by ‘spur’ formation immunodiffusion tests. Immunologically indistinguishable mercury reductases were found among strains belonging to phylogenetically distant genera. This suggests a horizontal transfer of mercury resistance genes between these strains.
Persistence of species variation and regional heterogeneity of the apparent molecular masses of benzodiazepine‐binding proteins after deglycosylation
Authors: Schmitz Elke , Friedl Waltraut , Reichelt Ralf , Hebebrand Johannes
Published: July 2002

Brain membrane preparations of different vertebrates were photoaffinity labeled with [3H]flunitrazepam and subsequently deglycosylated with endoglycosidase F and peptide N‐glycopeptidase. SDS‐polyacrylamide gel electrophoresis followed by fluorography revealed that each benzodiazepine‐binding protein is deglycosylated in two steps, indicating that each protein has two glycosylation sites. Species variation of the apparent molecular masses of the benzodiazepine‐binding proteins and regional heterogeneity in avians persist after deglycosylation. These results indicate that the α‐subunit(s) of the GABA/benzodiazepine receptor has undergone electrophoretically detectable changes in its amino acid composition during vertebrate evolution. The existence of at least two different α‐subunits in avians is further substantiated.
Dissociation of the ligand and dephosphorylation of the platelet‐derived growth factor receptor
Authors: Sturani E. , Zippel R. , Morello L. , Brambillo R. , Alberghina L.
Published: July 2002

The ligand‐induced phosphorylation of the platelet‐derived growth factor (PDGF) receptor was followed at 37°C by a rapid dephosphorylation which was roughly parallel to the down regulation of the 125I‐PDGF binding sites. At 4°C, when the ligand‐receptor complexes remain associated with the cell surface, the phosphorylated form of the receptor was more stable. However if the ligand was dissociated from the receptor by means of a mild acid wash or a treatment with suramin, the dephosphorylation of the receptor also occurred at a low temperature. These data suggest that, due to the dissociation of the ligand, the kinase activity of the receptor is switched off so that the phosphotyrosine‐containing receptors remain exposed to the action of phosphatases that rapidly dephosphorylate them.
Derivation of the sequence of the signal peptide in human C4b‐binding protein and interspecies cross‐hybridisation of the C4bp cDNA sequence
Authors: Lintin S.J. , Lewin A.R. , Reid K.B.M.
Published: July 2002

A 5′ cDNA clone coding for human C4b‐binding protein (C4bp) was isolated, characterised and sequenced to complete the cDNA sequence coding for residues 1–32 thus confirming the protein sequence data of Chung et al. [(1985) Biochem. J. 230, 133–141]. The sequence extended to allow derivation of the putative leader peptide sequence which was 32 residues in length and showed a high of hydrophobicity typical of other documented leader sequences. Cross hybridisation was detected between the human C4bp cDNA probes and genomic DNA isolated from various species on Southern blots suggesting that genomic sequence homologous to that coding for C4bp has been conserved during evolution.
Inactivation of tissue inhibitor of metalloproteinases by neutrophil elastase and other serine proteinases
Authors: Okada Yasunod , Watanabe Shoji , Nakanishi Isao , Kishi Jun-ichi , Hayakawa Taro , Watorek Wieslaw , Travis James , Nagase Hideaki
Published: July 2002

Tissue inhibitor of metalloproteinases (TIMP) from cultured bovine dental pulp inhibits human rheumatoid synovial matrix metalloproteinase 3 (MMP‐3) with a stoichiometry of 1:1 on a molar basis. Among the serine proteinases examined, human neutrophil elastase, trypsin and α‐chymotrypsin destroyed the inhibitory activity of TIMP against MMP‐3 by degrading the inhibitor molecule into small fragments. In contrast, the inhibitory activity of TIMP was not significantly reduced by the actions of cathepsin G, pancreatic elastase and plasmin. These data indicate that neutrophils which infiltrate tissues in various inflammatory conditions may play an important role in regulating TIMP activity in vivo through the action of neutrophil elastase.
Cooperative interactions of protein kinase C and cAMP‐dependent protein kinase systems in human promyelocytic leukemia HL60 cells
Authors: Deli Eva , Kiss Zoltan , Kuo J.F.
Published: July 2002

Interactions of protein kinase C (PKC) and cAMP‐dependent protein kinase (PKA) systems were investigated in HL60 cells. It was found that the differentiating effects of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) were potentiated by dibutyryl cAMP (dbcAMP) or prostaglandin E2 (PGE2). In addition, dbcAMP or PGE2 inhibited TPA‐induced binding of PKC to plasma membrane, leading to decreased protein phosphorylation, and promoted subsequent redistribution of enzyme to the nuclear membrane region. The findings are consistent with the hypothesis that PKC and PKA systems regulate cooperatively the phenotypical differentiation of leukemic cells.
Recombinant interferon‐β2 (interleukin‐6) induces myeloid differentiation
Authors: Chen Louisa , Novick Daniella , Rubinstein Menachem , Revel Michel
Published: July 2002

Human IFN‐β2 cytokine produced in E. coli was purified to homogeneity by immunoaffinity and ion‐exchange chromatography. The cytokine inhibits the growth of myeloleukemic M 1 cells and induces their morphological and functional differentiation into macrophages. Differentiation was also observed in the histiocytic lymphoma U937 cells. The effect on U937 was synergized by IFN‐γ and under these conditions IFN‐β2 produced the induction of (2′–5′) oligo(A) synthetase typical to IFN action and to differentiation.
Characterisation of the D1 protein in a photosystem II mutant (LF‐1) of Scenedesmus obliquus blocked on the oxidising side Evidence supporting non‐processing of D1 as the cause of the lesion
Authors: Taylor M.A. , Nixon P.J. , Todd C.M. , Barber J. , Bowyer J.R.
Published: July 2002

The D1 polypeptide of the photosystem II reaction centre in a mutant (LF‐1) of Scenedesmus obliquus lacking a water‐splitting manganese complex is approx. 1.5 kDa larger than that in the wild type but the D2 protein is the same size. The peptide profiles of D1 on partial digestion with papain or endoproteinase Lys‐C indicate that the extra segment in the LF‐1 protein is located at or near the carboxyl‐terminus. The D1 proteins produced by in vitro translation of mRNA from wild type and LF‐1 cells have an identical molecular mass to D1 from LF‐1 thylakoids whereas D1 from wild‐type thylakoids is 1.5 kDa smaller due to C‐terminal processing. These results support the hypothesis that in the LF‐1 mutant, the D1 protein is incorporated into the PS II reaction centre, but the C‐terminal extension is not removed.
Phosphorylation of neurofilament proteins by protein kinase C
Authors: Sihag Ram K. , Jeng Arco Y. , Nixon Ralph A.
Published: July 2002

The low molecular mass (70 kDa) subunit of neurofilaments (NF‐L) contains at least three phosphorylation sites in vivo and is phosphorylated by multiple kinases in a site‐specific manner [(1987) J. Neurochem. 48, S101; Sihag, R.K. and Nixon, R.A. submitted]. In this study, we observed that the three subunits of neurofilament proteins from retinal ganglion cell neurons are substrates for purified mouse brain protein kinase C. Two‐dimensional α‐chymotryptic phosphopeptide map analyses of the NF‐L subunit demonstrated that protein kinase C phosphorylates four polypeptide sites, two of which incorporate phosphate when retinal ganglion cells are pulse‐radiolabeled with [32P]orthophosphate in vivo.
Influence of substrate and template binding on the interaction of α‐amanitin with yeast RNA polymerase II: Fluorescence spectroscopic analysis
Authors: Bhargava Purnima , Chatterji Dipankar
Published: July 2002

Fluorescence titration of the tryptophan residues in yeast RNA polymerase II with α‐amanitin shows there are two types of binding between the inhibitor and the enzyme. When the stronger binding site was saturated with α‐amanitin, the enzyme‐inhibitor complex could not be further titrated efficiently with ATP, indicating that probably the inhibitor and the substrate binding domains on the enzyme are overlapping. Supercoiled plasmid DNA bearing alcohol dehydrogenase I promoter of yeast showed binding with purified yeast RNA polymerase II in the absence of substrates. However, when the enzyme‐inhibitor complex was titrated with this template, it was found that the complex behaves in a similar way as the enzyme alone towards the template DNA. It suggests that probably the inhibitor and the template binding sites on the enzyme are quite different.
Cleavages of aromatic ring and β‐O‐4 bond of synthetic lignin (DHP) by lignin peroxidase
Authors: Umezawa Toshiaki , Higuchi Takayoshi
Published: July 2002

Lignin peroxidase from a white‐rot basidiomycete, Phanerochaete chrysosporium, catalyzed cleavages of the aromatic ring and the β‐O‐4 bond of a synthetic lignin, a dehydrogenation copolymer (DHP) of coniferyl alcohol and a (β‐O‐4)‐(β‐β) lignin substructure model trimer.
Photolabeled tryptic degradation products of benzodiazepine‐binding proteins are glycopeptides Implications for localization of cleavage sites
Authors: Schmitz Elke , Reichelt Ralf , Möhler Hanns , Hebebrand Johannes
Published: July 2002

Crude synaptic membranes of avian and mammalian brain tissue were photolabeled with the benzodiazepine‐receptor ligand [3H]flunitrazepam and subsequently treated extensively with trypsin followed by incubation with endoglycosidase F. SDS‐polyacrylamide gel electrophoresis and fluorography revealed that the final tryptic degradation product of 25 kDa in both pigeon and calf brain is deglycosylated in two steps. These results were confirmed by immunoblots of similarly pretreated membranes of pig brain using the α‐subunit‐specific monoclonal antibody bd‐24. Benzodiazepine‐receptor binding and its enhancement by GABA are largely retained after trypsinization. Based on the proposed transmembrane topology for the α‐subunits of the GABA/benzodiazepine receptor, we suggest that the large N‐terminal domain of benzodiazepine‐binding proteins is protected against tryptic cleavage.
Immunological evidence for a relationship between the dendrotoxin‐binding protein and the mammalian homologue of the Drosophila Shaker K+ channel
Authors: Rehm Hubert , Newitt Richard A. , Tempel Bruce L.
Published: July 2002

Polyclonal antibodies were raised against two synthetic peptides from different parts of the predicted amino acid sequence of the mouse homologue (MBK1) of the Drosophila Shaker K+ channel. The antibodies recognized the toxin‐binding subunit of the dendrotoxin‐binding proteins from rat and bovine brain. The results suggest that the dendrotoxin‐binding protein is related to the expression products of the mammalian homologue of the Shaker gene.
Identification of thiol groups and a disulfide crosslink site in bovine myelin proteolipid protein
Authors: Shaw Shyh-Yu , Laursen Richard A. , Lees Marjorie B.
Published: July 2002

The existence of disulfide crosslinks limits the number of possible folded structures a protein can assume. Thus localization of disulfide and thiol groups is a key to understanding the conformation and orientation of myelin proteolipid protein (PLP) in the myelin membrane. [14C]Carboxamidomethylated PLP was fragmented with chymotrypsin, and the resulting mixture was partially separated by reversed‐phase HPLC. Purified 14C‐labeled peptides and a disulfide containing peptide were characterized by amino acid analysis. These experiments showed that Cys‐32 and Cys‐34 are free thiols, and are presumably on the interior of the cell or within the membrane bilayer, and that Cys‐200 and Cys‐219 are joined by a disulfide bond, and are probably located on the extracellular face of the membrane. Sequence analysis experiments indicate that Cys‐5, Cys‐6 and Cys‐9 are linked by disulfides, probably to other parts of the protein on the extracellular face of the membrane.
Site‐selective 8‐C1‐cAMP which causes growth inhibition and differentiation increases DNA (CRE)‐binding activity in cancer cells
Authors: Mednieks Maija I. , Yokozaki Hiroshi , Merlo Giorgio R. , Tortora Giampaolo , Clair Timothy , Ally Shamsia , Tahara Eiichi , Cho-Chung Yoon Sang
Published: July 2002

Control mechanisms of normal differentiation are disrupted in cancer cells but can be restored by treatment with site‐selective cAMP analogs. The cellular events associated with such changes entail compartmental redistribution of the cAMP‐dependent protein kinase type II regulatory subunit, RIIβ. The results of this study indicate that the molecular mechanisms of action involve changes in specific DNA‐binding activity of putative transcription factors. Gel retardation analyses revealed that nuclear extracts from cells of various human cancer cell lines [colon cancer (LS‐174T), gastric cancer (TMK‐1), and leukemia (K‐562)] and rodent pheochromocytoma (PC12) show a concentration‐dependent increase in binding activity to a synthetic DNA that contained the cAMP‐responsive element 5′‐TGACGTCA‐3′ after treatment with 8‐C1‐cAMP. Such an increase in cAMP‐responsive element binding activity was not observed in the 8‐C1‐cAMP‐unresponsive MKN‐1 gastric cancer cells. These findings indicate that the antitumor activity of site‐selective cAMP analogs may reside in the induction of transcription factors that restore normal gene regulation in cancer cells.
Isozymic forms of protein kinase C in regenerating rat liver
Authors: Houweling M. , Vaartjes W.J. , van Golde L.M.G.
Published: July 2002

The expression of multiple forms of protein kinase C (PK‐C) was studied in regenerating rat liver using hydroxyapatite column chromatography. Two forms of the enzyme were found in the cytosolic as well as membrane fraction of livers from partially hepatectomized rats. The kinetic variation in the activation of these two liver isozymes by fatty acids, phosphatidylserine and diacylglycerol was similar to that reported for the PK‐C subspecies from rat brain, designated types II and III. Intracellular redistribution of PK‐C caused by phorbol 12‐myristate 13‐acetate (PMA) was concentration‐dependent and was due to translocation of isozyme III, because type II was insensitive to 5 × 10−8 M PMA. The activity ratio of the two isozymes in either the particulate or cytosolic fraction was the same at 22 h as compared to 4 h after partial hepatectomy.
A patch‐clamp study of ion channels of inner and outer membranes and of contact zones of E. coli, fused into giant liposomes
Authors: Berrier Catherine , Coulombe Alain , Houssin Christine , Ghazi Alexandre
Published: July 2002

Inner and outer membranes of Escherichia coli and contact zones were isolated and fused separately with giant liposomes amenable to patch‐clamp recording. Different types of large pressure‐activated channels were localized in the inner membrane fraction which also contained smaller, pressure‐insensitive channels. The outer membrane contained pressure‐insensitive channels with large conductances and long opening and closing times which are likely to be porins. Large channels were also observed in contact zones.