bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural relationship between the mammalian Fe(III)‐Fe(II) and the Fe(III)‐Zn(II) plant purple acid phosphatases
Authors: Klabunde Thomas , Sträter Norbert , Krebs Bernt , Witzel Herbert
Published: May 2003

The primary structure of uteroferrin (Uf), a 35 kDa monomeric mammalian purple acid phosphatase (PAP) containing a Fe(III)‐Fe(II) center, has been compared with the sequence of the homodimeric 111 kDa Fe(III)‐Zn(II) kidney bean purple acid phosphatase (KBPAP). The alignment suggests that the amino acid residues ligating the dimetal center are identical in Uf and KBPAP, although the geometry of the coordination sphere might slightly differ. Secondary structure predictions indicate that Uf contains two βαβαβ motifs thus resembling the folding topology of the plant enzyme. Guided by the recently determined X‐ray structure of KBPAP a tentative model for the mammalian PAP can be constructed.
Calf spleen purine nucleoside phosphorylase: purification, sequence and crystal structure of its complex with an N(7)‐acycloguanosine inhibitor
Authors: Bzowska Agnieszka , Luić Marija , Schröder Werner , Shugar David , Saenger Wolfram , Koellner Gertraud
Published: May 2003

Calf spleen purine nucleoside phosphorylase was purified to homogeneity and its amino acid sequence was determined. The complex of the enzyme with an N(7)‐acycloguanosine inhibitor crystallized in the cubic space group P213, with unit cell dimension and one monomer in the asymmetric crystal unit. The biologically active trimer is formed by the crystallographic three‐fold axis. The structure was solved by molecular replacement methods, using the model of the human erythrocyte enzyme, and refined at a resolution of 2.9 Å to an R‐factor of 0.21. The orientation of the inhibitor at the active site is examined in relation to the catalytic activity of the enzyme in the phosphorolysis of nucleosides.
Microvesicles isolated from bovine pineal gland specifically accumulate l‐glutamate
Authors: Moriyama Yoshinori , Yamamoto Akitsugu
Published: May 2003

Pinealocytes, endocrine cells that synthesize and secrete melatonin, possess a large number of synaptic‐like microvesicles (MVs) containing synaptophysin. By monitoring cross‐reactivity with anti‐synaptophysin antibody, the MVs were highly purified from bovine pineal glands. The purified MVs were morphologically similar to but distinct from neuronal synaptic vesicles by their lack of synapsin I. Immunological study indicated that the MVs contained vacuolar H+‐ATPase, synaptotagmin and synaptobrevin 2 (VAMP2). The MVs accumulated l‐glutamate at the expense of ATP hydrolysis by vacuolar H+‐ATPase. No uptakes of melatonin, serotonin, noradrenaline, γ‐aminobutyrate or acetylcholine were observed. These results indicated that the MVs are organelles for storage of l‐glutamate in pinealocytes and suggested a possibility that pinealocytes transmit glutamate signals by MVs‐mediated exocytosis.
N,N,N‐trimethylsphingosine inhibits interleukin‐1β‐induced NF‐κB activation and consequent E‐selectin expression in human umbilical vein endothelial cells
Authors: Masamune Atsushi , Hakomori Sen-itiroh , Igarashi Yasuyuki
Published: May 2003

We examined the effect of N,N,N‐trimethylsphingosine (TMS) on the interleukin‐1β (IL‐1β)‐induced E‐selectin expression in human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with TMS (0.1–10 μM) resulted in a concentration‐dependent inhibition of IL‐1β‐induced E‐selectin expression. Sphingosine or N,N‐dimethylsphingosine had no effects on the expression. Electrophoretic mobility shift assay revealed that TMS inhibited IL‐1β‐induced NF‐κB activation, which is essential for E‐selectin expression. This inhibitory effect of TMS on IL‐1β‐dependent endothelial cell activation may partly explain the known anti‐inflammatory or anti‐metastatic effect of TMS in vivo.
The mRNA‐binding protein COLBP is glutamate dehydrogenase
Authors: Preiss Thomas , Sang A.Elizabeth , Chrzanowska-Lightowlers Zofia M.A. , Lightowlers Robert N.
Published: May 2003

Expression of the liver‐type isopeptides of cytochrome c oxidase is regulated post‐transcriptionally. An RNA‐binding activity has been found in only those cells where the liver‐type subunits are detected. This binding protein, termed COLBP, recognises sequences or structures within the 3′‐untranslated regions of transcripts encoding these liver‐type isopeptides and has been implicated in the modulation of mRNA expression. We now show by subcellular fractionation, immunocompetition, UV‐crosslinking and shift‐Western studies that the metabolic enzyme glutamate dehydrogenase, previously reported as being able to bind RNA, is the cytochrome c oxidase transcript‐binding protein, COLBP.
Involvement of rho in GTPγS‐induced enhancement of phosphorylation of 20 kDa myosin light chain in vascular smooth muscle cells: inhibition of phosphatase activity
Authors: Noda Masakuni , Yasuda-Fukazawa Chikako , Moriishi Kohji , Kato Tetsuo , Okuda Toshihiro , Kurokawa Kiyoshi , Takuwa Yoh
Published: May 2003

In β‐escin‐permeabilized cultured pig aortic smooth muscle cells GTPγS dose‐dependently enhances Ca2+‐induced wortmannin‐sensitive phosphorylation of 20 kDa myosin light chain (MLC20). GTPγS does not potentiate thiophosphorylation of MLC20, but does inhibit its dephosphorylation. Pretreatment with C. botulinum exotoxin C3, which specifically ADP‐ribosylates and inactivates the rho family of the small molecular weight G proteins, completely abolishes the effects of GTPγS. These results indicate that rho is involved in the GTPγS‐induced enhancement of Ca2+‐dependent MLC20 phosphorylation in aortic smooth muscle cells, and strongly suggest that this effect of rho is due to inhibition of protein phosphatase activity toward MLC20.
Cyclin‐dependent kinases and pRb: regulators of the proliferation‐differentiation switch
Authors: Kranenburg O. , van der Eb A.J. , Zantema A.
Published: May 2003

The retinoblastoma susceptibility gene (RB1) is essential for normal embryonic development. Loss of RB1 leads to uncontrolled proliferation of a number of cell types but may also prevent proper terminal differentiation. The growth‐suppressive and differentiation‐inducing properties of pRb are impaired by cyclin‐dependent kinase (cdk)‐mediated phosphorylation. Hence, inhibition of cdk activity is probably a prerequisite for terminal differentiation. Indeed, forced cyclin or cdk expression can prevent terminal differentiation in various cell types, probably through inhibition of pRb and, possibly, differentiation‐specific transcription factors.
Comparisons of P‐glycoprotein expression in isolated rat brain microvessels and in primary cultures of endothelial cells derived from microvasculature of rat brain, epididymal fat pad and from aorta
Authors: Barrand M.A. , Robertson K.J. , von Weikersthal S.F.
Published: May 2003

In vivo expression of P‐glycoprotein in isolated rat brain microvessels is compared with that in vitro in primary cultures of brain endothelial cells. More P‐glycoprotein is detected by Western immunoblotting in microvessels than in cultured endothelium. RT‐PCR with isoform‐specific primers and immunoblotting with a mdr1b‐specific antibody reveals only mdr1a in vivo but both mdr1a and mdr1b in vitro. Thus mdr1a decreases whereas mdr1b increases during culture. P‐Glycoprotein activity is evident in vitro, with resistance modulators, e.g. verapamil, producing increases in intracellular [3H]vincristine accumulation. Endothelial cells cultured from epididymal fat pad microvasculature and aorta contain little or no P‐glycoprotein. Here, resistance modulators are less effective.
In vivo inactivation of phosphotyrosine protein phosphatases by nitric oxide
Authors: Caselli Anna , Chiarugi Paola , Camici Guido , Manao Giampaolo , Ramponi Giampietro
Published: May 2003

The effect of NO on phosphotyrosine protein phosphatases (PTPases) has been investigated in vivo. NO production is induced in interferon‐γ and lipopolyaccharide stimulated RAW‐264.7 macrophages as indicated by the increase of NO2
− in the medium. Our results demonstrate an inhibition of p‐nitrophenylphosphatase activity as a consequence of macrophages activation. Under the described experimental conditions, most of the hydrolysis of p‐nitrophenylphosphate can be ascribed to the action of cellular PTPases. The presence of , a specific inhibitor of NO synthase decreases the inactivation rate of both membrane‐bound and soluble PTPases. This evidence further confirms the ability of NO to inactivate PTPases and suggests a possible role of NO in the regulation of cellular processes involving this class of phosphatases.
Protein O‐glycosylation in Saccharomyces cerevisiae: the protein O‐mannosyltransferases Pmt1p and Pmt2p function as heterodimer
Authors: Gentzsch Martina , Immervoll Thomas , Tanner Widmar
Published: May 2003

The protein O‐mannosyltransferases Pmt1p and Pmt2p are catalyzing the O‐glycosylation of serine and threonine residues in the endoplasmic reticulum of yeast. Deletion of each of these proteins by disruption of the corresponding gene leads to a dramatic decrease of mannosyltransferase activity in vitro. With an anti‐Pmt1p immunoaffinity column a complex of Pmt1p and a second protein was purified; this protein turned out to be Pmt2p. Overexpression of Pmt1p or Pmt2p, respectively, does not increase mannosyltransferase activity in vitro. Overexpression of both mannosyltransferases together, however, raises in vitro activity threefold. These data indicate that Pmt1p and Pmt2p function as a complex catalyzing protein O‐glycosylation in yeast.
The human β2‐adrenergic receptor expressed in Schizosaccharomyces pombe retains its pharmacological properties
Authors: Ficca Anna Grazia , Testa Luana , Tocchini Valentini Glauco P.
Published: May 2003

We have developed a rapid and efficient expression system to study the human β
2 adrenergic receptor (huβ
2AR) in the fission yeast Schizosaccharomyces pombe. This was achieved by cloning the huβ
2AR gene, modified by replacement of the 5′ untranslated and a small part of the N‐terminal coding sequence (first 14 amino acids) with the corresponding region of the yeast Saccharomyces cerevisiae STE2 (α‐factor receptor) gene. The gene was then placed under the control of a S. pombe constitutive promoter for alcohol dehydrogenase (adh). Huβ
2AR expression was assessed by immunoblot analysis of the chimeric protein with an anti‐STE2 serum raised against a dodecapeptide homologous to the N‐terminal amino acids of STE2 and ligand binding was assayed using [125I]cyanopindolol. We demonstrate here that the chimeric receptor expressed in S. pombe exhibits the same characteristic ligand specificity and affinity as that of the authentic huβ
2AR. This system constitutes a convenient alternative to existing methods for studying seven transmembrane domain receptors due to its simplicity and high reproducibility.
Evidence against specific binding of salicylic acid to plant catalase
Authors: Rüffer Martina , Steipe Boris , Zenk Meinhart H.
Published: May 2003

It was demonstrated that salicylic acid (SA) not only binds to catalase from differentiated higher plants and plant cell suspension cultures but also to those of fungi and animals. SA bound specifically to iron‐containing enzymes, such as catalase, aconitase, lipoxidase and peroxidase, while not to iron‐free plant enzymes. On the grounds of these experiments, the claim is further challenged that SA is a signalling compound and second messenger in plants that activates plant defense‐related genes through elevated H2O2 levels by specifically inhibiting catalase activity. SA may just function as a phytoalexin.
The pharmacological profile of the vesicular monoamine transporter resembles that of multidrug transporters
Authors: Yelin Rodrigo , Schuldiner Shimon
Published: May 2003

Vesicular neurotransmitter transporters function in synaptic vesicles and other subcellular organelles and they were thought to be involved only in neurotransmitter storage. Several findings have led us to test novel aspects of their function. Cells expressing a c‐DNA coding for one of the rat monoamine transporters (VMAT1) become resistant to the neurotoxin N‐methyl‐4‐phenylpyridinium (MPP+) [Liu et al. (1992) Cell, 70, 539–551]. The basis of the resistance is the VMAT1‐mediated transport and sequestration of the toxin into subcellular compartments. In addition, the deduced sequence of VMAT1 predicts a protein that shows a distinct homology to a class of bacterial drug resistance transporters (TEXANs) that share some substrates with mammalian multidrug resistance transporters (MDR) such as the P‐glycoprotein. These findings induced us to test whether compounds that are typically transported by MDR interact also with vesicular transporters. The use of [3H]reserpine binding to determine drug interactions with VMAT allowed assessment of the ability of various drugs to bind to the substrate site of the transporter. Cytotoxic compounds such as ethidium, isometamidium, tetraphenylphosphonium, rhodamine, tacrine and doxorubicin, interact specifically with vesicular monoamine transporters. Verapamil, a calcium channel blocker, is also a competitive inhibitor of transport. In the case of rhodamine, fluorescence measurements in digitonin‐permeabilized cells demonstrated ATP‐dependent VMAT‐mediated transport. The results imply that even though the bacterial and vesicular transporters are structurally different from the P‐glycoprotein, they share a similar substrate range. These findings suggest a novel possible way of protection from the effects of toxic compounds by removal to subcellular compartments.
Human ClpP protease: cDNA sequence, tissue‐specific expression and chromosomal assignment of the gene
Authors: Bross Peter , Andresen Brage S. , Knudsen Inga , Kruse Torben A. , Gregersen Niels
Published: May 2003

We identified three overlapping human expressed sequence tags with significant homology to the E. coli ClpP amino sequence by screening the EMBL nucleotide database. With this sequence information we applied 5′ and 3′‐rapid amplification of cDNA ends (RACE) to amplify and sequence human clpP cDNA in two overlapping fragments. The open reading frame encodes a 277 amino acid long precursor polypeptide. Two ClpP specific motifs surrounding the active site residues are present and extensive homology to ClpP's from other organisms was observed. Northern blotting showed high relative expression levels of clpP mRNA in skeletal muscle, intermediate levels in heart, liver and pancreas, and low levels in brain, placenta, lung and kidney. By analysis of human/rodent cell hybrids the human clpP gene was assigned to chromosome 19.
Identification of the nicking tyrosine of geminivirus Rep protein
Authors: Laufs Jürgen , Schumacher Silke , Geisler Norbert , Jupin Isabelle , Gronenborn Bruno
Published: May 2003

The replication initiator (Rep) proteins of geminiviruses perform a DNA cleavage and strand transfer reaction at the viral origin of replication. As a reaction intermediate, Rep proteins become covalently linked to the 5′ end of the cleaved DNA. We have used tomato yellow leaf curl virus Rep protein for in vivo and in vitro analyses. Isolating a covalent peptide‐nucleotide complex, we have identified the amino acid of Rep which mediates cleavage and links the protein to DNA. We show that tyrosine‐103, located in a conserved sequence motif, initiates DNA cleavage and is the physical link between geminivirus Rep protein and its origin DNA.
Functional expression of bacteriorhodopsin in oocytes allows direct measurement of voltage dependence of light induced H+ pumping
Authors: Nagel Georg , Möckel Bettina , Büldt Georg , Bamberg Ernst
Published: May 2003

We report on the first successful expression of the light driven H+ pump, bacteriorhodopsin, into the plasma membrane of oocytes from Xenopus laevis. The light induced photocurrents which reflect the pumping of H+ by BR were analysed under voltage clamp conditions. At least 100 active BR molecules per μm2 were expressed in the plasma membrane so that both the voltage clamp and giant patch clamp method could be applied. We show that H+ pumping by BR is modulated by the membrane potential, i.e. the pump current shows strong voltage dependence in the range measured between −165 mV to +60 mV.
Constitutive activity of the M1–M4 subtypes of muscarinic receptors in transfected CHO cells and of muscarinic receptors in the heart cells revealed by negative antagonists
Authors: Jakubík Jan , Bačáková Lucie , El-Fakahany Esam E. , Tuček Stanislav
Published: May 2003

We investigated whether muscarinic receptors of the M1–M4 receptor subtypes are constitutively active. We have found that the synthesis of cyclic AMP was enhanced by the muscarinic antagonists atropine and N‐methylscopolamine (NMS) in Chinese hamster ovary (CHO) cells stably transfected with human m2 and m4 muscarinic receptor genes and in rat cardiomyocytes expressing the M2 receptor subtype, and that the production of inositol phosphates was inhibited by atropine and NMS in CHO cells stably transfected with human m1 and m3 and with rat m1 muscarinic receptor genes. The muscarinic antagonists quinuclidinyl benzilate and AF‐DX 116 had no effect in some cases and acted like atropine and NMS in others. We conclude that the M1–M4 subtypes of muscarinic receptors are constitutively active in the CHO cell lines expressing them and in cardiomyocytes and that atropine and NMS act as negative antagonists on these receptor subtypes by stabilizing them in the inactive conformation.
Alpha‐crystallin acting as a molecular chaperone protects catalase against steroid‐induced inactivation
Authors: Hook Darren W.A. , Harding John J.
Published: May 2003

A link between corticosteroid therapy and the development of cataract has been known for many years. However, the precise underlying molecular mechanism of pathology has not been characterised, although a role for direct deleterious interactions between corticosteroids and lenticular proteins has been investigated. α‐Crystallin is a major lens protein that has exhibited chaperone properties in vitro. Catalase is a ubiquitous enzyme that is an important scavenger of hydrogen peroxide in vivo. The corticosteroid prednisolone‐21‐hemisuccinate was found to inactivate bovine liver catalase, in vitro in a progressive manner. Coincubation of α‐crystallin with catalase in a 1:2 molar ratio (one α‐crystallin to two catalase molecules) fully protected against this inactivation. The protection was specific. Aspirin‐like analgesics, putative anti‐cataract drugs offered no such protection.
The presence of a glucose‐derived Maillard reaction product in the human lens
Authors: Nagaraj Ramanakoppa H. , Sady Candace
Published: May 2003

Pyrraline is an advanced Maillard reaction product formed by the non‐enzymatic reaction initiated by glucose with lysine residues on proteins. This reaction involves an intermediate, 3‐deoxyglucosone, concentration of which is shown to be elevated in plasma and lenses during diabetes. Bovine lens alpha crystallins incubated with 3‐deoxyglucosone showed that pyrraline formation was a major modification and its quantification by two different methods revealed time‐dependent accumulation. Pyrraline was quantified in normal, senile cataractous and diabetic lenses. Although a wide variation was observed, the mean value in cataractous lenses (mean ± S.E.: 48.4 ± 12.67 pmol/mg protein) was higher than in age‐matched normal lenses (30.9 ± 10.26 pmol). Surprisingly, in diabetic lenses, the mean value was lower than normal lenses (28.4 ± 15.3 pmol). These results suggest that glucose‐specific advanced Maillard products occur in the human lens and such modifications may play a role in lens aging and cataract formation.
Inversion of configuration during hydrolysis of α‐1,4‐galacturonidic linkage by three Aspergillus polygalacturonases
Authors: Biely P. , Benen J. , Heinrichová K. , Kester H.C.M. , Visser J.
Published: May 2003

Endopolygalacturonases I and II (PGI and PGII) of Aspergillus niger and an exopolygalacturonase (ExoPG) of A. tubingensis were investigated to reveal the stereochemistry of their hydrolytic action. Reduced pentagalacturonic acid (pentaGalU‐ol) and reduced trigalacturonic acid (triGalU‐ol) were used as non‐reducing substrates for the enzymes. The configuration of the reducing ends in the products formed in D2O reaction mixtures was followed by 1H‐NMR spectroscopy. It has been unambiguously established that primary cleavage of pentaGalU‐ol by both PGI and PGII leads to diGalU‐ol and the β‐anomer of triGalUA. The primary products of hydrolysis of triGalU‐ol by ExoPG were diGalU‐ol and the β‐anomer of GalUA. Thus, all three Aspergillus polygalacturonases belong to the so‐called inverting glycanases, i.e. they utilize the single displacement mechanism of hydrolysis of the glycosidic linkage.
Activities associated with the presence of ribosome‐inactivating proteins increase in senescent and stressed leaves
Authors: Stirpe Fiorenzo , Barbieri Luigi , Gorini Paola , Valbonesi Paola , Bolognesi Andrea , Polito Letizia
Published: May 2003

The ribosome‐inactivating proteins (RIPs) from Hura crepitans and Phytolacca americana release adenine from herring sperm DNA. Leaf extracts from these plants show the same enzymatic activities as the RIPs. The translation inhibitory activity and the activity on DNA are both increased in the leaves of both plants during senescence or when subjected to heat or osmotic stress. It is proposed that a physiological role of RIPs could be to intervene in the death of plant cells.
Service markets and competition
Authors: Karmarkar Uday S. , Pitbladdo Richard
Published: May 2003

The production and delivery of services differ from manufacturing in ways which have a significant impact on the nature of market competition. Contracts for manufactured goods are centered around a clearly defined junction between production and use, made possible by the properties of tangibility and portability of tangible goods, at which point responsibility for use, operation and consumption of the product is transferred from producer to customer, along with ownership. Service operations, by contrast, involve a lack of inventories and portability, customer contact, joint production, customer‐specific inputs, and intangibility in varying degrees. Moreover, service operations are either entirely post contractual or interlinked with the contracting process. As a result, the very nature of service markets and contracting depends on the characteristics of the service operation. While the operational consequences of service characteristics have been explored quite widely, there is less understanding of the special features of service competition and its implications for service strategy. We provide a framework for describing, analyzing and explaining service competition and review some old and new models which address the special features of service markets.
Economics of total quality management
Authors: Lederer Phillip J. , Rhee Seung‐Kyu
Published: May 2003

This paper presents two models of the economics of total quality management. In the first, the concept of quality management is viewed as a technological innovation that requires investment. To reduce cost and improve quality, firms must make investments that are largely sunk. The effect of market competition on quality related technology investments is studied. Several results follow. With new quality technologies, price falls, quality rises and average cost declines. Firms must anticipate rivals' technology choices and the market prices when justifying quality technology investments. When all firms quickly adopt quality technology, returns of such investments are normal, that is, have a zero net present value. However, firms that do not invest in quality related technology are forced from the market. A firm that is faced by competitors that are slow to adopt quality related technology, can earn positive returns by early adoption. The firm invests more in quality related technology, and produces higher quality products, charges a higher price and earns higher profits than competitors. The firm's quality, price and profit advantages persist over time. In the second model, we show that firm value increases when customer satisfaction is used as an objective by aligning incentives. This explains the common use of customer satisfaction measures in TQM programs.
Hierarchical capacity expansion and production planning decisions in stochastic manufacturing systems
Authors: Sethi S.P. , Taksar M. , Zhang Q.
Published: May 2003

We present an approach of hierarchical decision making in production planning and capacity expansion problems under uncertainty. We show that under reasonable assumptions, the strategic level management can base the capacity decision on aggregated information from the shopfloor, and the operational level management, given this decision, can derive a production plan for the system, without too large a loss in optimality when compared to simultaneous determination of optimal capacity and production decisions.

The results are obtained via an asymptotic analysis of hierarchical investment and production decisions in a manufacturing system with machines subject to breakdown and repair. The demand facing the system is assumed to be a deterministic monotone increasing function. The production capacity can be increased by purchasing a finite number of new machines over time. The control variables are a sequence of purchasing times and a production plan. The rate of change in machine states is assumed to be much larger than the rate of discounting of costs. This gives rise to a limiting problem in which the stochastic machine availability is replaced by the equilibrium mean availability. The value function for the original problem converges to the value function of the limiting problem. Three different methods are developed for constructing decisions for the original problem from the optimal solution of the limiting problem in a way which guarantees the asymptotic optimality of constructed decisions. Finally, it is shown that as the number of machine that could be purchased tends to infinity, the problem approximates the corresponding problem with no limit on number of machine purchases.
Simultaneous determination of product attributes and prices, and production processes in product‐line design
Authors: Raman Narayan , Chhajed Dilip
Published: May 2003

Global competition, rapid changes in technology, and market fragmentation have resulted in shorter product life cycles. In order to remain viable, it is increasingly important for firms to introduce new products frequently. Product design is a complex process that involves coordination of activities among several functional disciplines within the company as well as the customers and the suppliers. Traditionally, the information flow among the various product development stages has been sequential. However, there is increasing evidence to suggest that an integrated approach that considers several stages simultaneously may be superior.

This paper provides a decision support tool for implementing such an integrated approach. On the basis of given customer preferences, the paper presents a model for determining the number of new products to be introduced, the exact specifications of these products, and the production processes for efficiently delivering these specifications. These decisions are made in an integrated manner by simultaneously considering the interaction among the various choice variables. A decomposition‐based solution procedure is developed that iterates between the product design and process selection decisions while maintaining an effective link between them.

In addition to understanding the economic value of adopting the integrated approach to product design, the paper discusses how the proposed model can be used effectively to perform sensitivity analysis with respect to some of the important decision variables.
Identification and characterisation of human aldose 1‐epimerase
Authors: Timson David J. , Reece Richard J.
Published: May 2003

Aldose 1‐epimerase or mutarotase (EC 5.1.3.3) is a key enzyme of carbohydrate metabolism catalysing the interconversion of the α‐ and β‐anomers of hexose sugars such as glucose and galactose. We identified an open reading frame in the human genome (BC014916) which has high sequence similarity to previously identified bacterial aldose 1‐epimerases. This sequence was cloned into a bacterial expression vector, and expressed and purified from this source. Enzyme assays show that the protein has aldose 1‐epimerase activity and exhibits a preference for galactose over glucose. Site‐directed mutagenesis confirmed the involvement of three residues involved in catalysis and substrate binding.
Identification of two serine residues important for p53 DNA binding and protein stability
Authors: Wei Gang , Liu Guoxia , Liu Xuan
Published: May 2003

The p53 core DNA binding domain has been implied in Mdm2‐mediated protein degradation. Here we show that the substitution of the serine residues 116 and 127 with alanine residues (S116/127A) has no effect on p53 DNA binding and protein stability. However, the substitution of the serine residues with the aspartic acid (S116/127D) abolished p53 DNA binding and led to protein stabilization. Importantly, we have shown that S116/127D exhibits a structural mutant conformation that results in a loss of p53‐dependent transcription and Mdm2‐mediated protein degradation.
Protonmotive pathways and mechanisms in the cytochrome bc1 complex
Authors: Hunte Carola , Palsdottir Hildur , Trumpower Bernard L
Published: May 2003

The cytochrome bc
1 complex catalyzes electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which electron transfer is linked to proton translocation across the inner mitochondrial membrane. In the Q cycle mechanism proton translocation is the net result of topographically segregated reduction of quinone and reoxidation of quinol on opposite sides of the membrane, with protons being carried across the membrane as hydrogens on the quinol. The linkage of proton chemistry to electron transfer during quinol oxidation and quinone reduction requires pathways for moving protons to and from the aqueous phase and the hydrophobic environment in which the quinol and quinone redox reactions occur. Crystal structures of the mitochondrial cytochrome bc
1 complexes in various conformations allow insight into possible proton conduction pathways. In this review we discuss pathways for proton conduction linked to ubiquinone redox reactions with particular reference to recently determined structures of the yeast bc
1 complex.
Chemical structure and immunobiological activity of lipid A from Prevotella intermedia ATCC 25611 lipopolysaccharide
Authors: Hashimoto Masahito , Asai Yasuyuki , Tamai Riyoko , Jinno Takayoshi , Umatani Katsumi , Ogawa Tomohiko
Published: May 2003

The novel chemical structure and immunobiological activities of Prevotella intermedia ATCC 25611 lipid A were investigated. A lipopolysaccharide (LPS) preparation of P. intermedia was extracted using a phenol–chloroform–petroleum ether method, after which its purified lipid A was prepared by weak acid hydrolysis followed by chromatographic separations. The lipid A structure was determined by mass spectrometry and nuclear magnetic resonance to be a diglucosamine backbone with a phosphate at the 4‐position of the non‐reducing side sugar, as well as five fatty acids containing branched long chains. It was similar to that of Bacteroides fragilis and Porphyromonas gingivalis, except for the phosphorylation site. P. intermedia lipid A induced weaker cytokine production and NF‐κB activation in murine cells via Toll‐like receptor (TLR) 4 as compared to Escherichia coli synthetic lipid A (compound 506). Our results indicate that P. intermedia lipid A activates cells through a TLR4‐dependent pathway similar to E. coli‐type lipid A, even though these have structural differences.
Initiation factor eIF2B not p70 S6 kinase is involved in the activation of the PI‐3K signalling pathway induced by the v‐src oncogene
Authors: Vojtěchová Martina , Šloncová Eva , Kučerová Dana , Jiřička Jaroslav , Sovová Vlasta , Tuháčková Zdena
Published: May 2003

Our data show that in hamster fibroblasts transformed by Rous sarcoma virus (RSV), the phosphoinositide 3′‐kinase (PI‐3K)/Akt/glycogen synthase kinase 3 antiapoptotic pathway is upregulated and involved in increased protein synthesis through activation of initiation factor eIF2B. Upon inhibition of PI‐3K by wortmannin, phosphorylation of 70‐kDa ribosomal protein S6 kinase (p70 S6k) and its physiological substrate, ribosomal protein S6, decreased in the non‐transformed cells but not in RSV‐transformed cells. Thus PI‐3K, which is thought to be involved in regulation of p70 S6k, signals to p70 S6k in normal fibroblasts, but it does not appear to be an upstream effector of p70 S6k in fibroblasts transformed by v‐src oncogene, suggesting that changes in the PI‐3K signalling pathway upstream of p70 S6k are induced by RSV transformation.
Interaction of bilirubin and biliverdin with reactive nitrogen species
Authors: Kaur Harparkash , Hughes Martin N. , Green Colin J. , Naughton Patrick , Foresti Roberta , Motterlini Roberto
Published: May 2003

Bilirubin (BR) and biliverdin (BV), two metabolites produced during haem degradation by haem oxygenase, possess strong antioxidant activities toward peroxyl radical, hydroxyl radical and hydrogen peroxide. Considering the importance attributed to nitric oxide (NO) and its congeners in the control of physiological and pathophysiological processes, we examined the interaction of BR and BV with NO and NO‐related species in vitro. Exposure of BR and BV to agents that release NO or nitroxyl resulted in a concentration‐ and time‐dependent loss of BR and BV, as assessed by high performance liquid chromatography. Peroxynitrite, a strong oxidant derived from the reaction of NO with superoxide anion, also showed high reactivity toward BR and BV. The extent of BR and BV consumption largely depended on the NO species being analysed and on the half‐lives of the pharmacological compounds considered. Of major importance, BR and BV decomposition occurred also in the presence of pure NO under anaerobic conditions, confirming the ability of bile pigments to scavenge the gaseous free radical. Increasing concentrations of thiols prevented BR consumption by nitroxyl, indicating that bile pigments and thiol groups can compete and/or synergise the cellular defence against NO‐related species. In view of the high inducibility of haem oxygenase‐1 by NO‐releasing agents in different cell types, the present findings highlight novel anti‐nitrosative characteristics of BR and BV suggesting a potential function for bile pigments against the damaging effects of uncontrolled NO production.
EPR study of light‐induced regulation of photosynthetic electron transport inSynechocystissp. strain PCC 6803
Authors: Trubitsin Boris V , Mamedov Mahir D , Vitukhnovskaya Liya A , Semenov Alexey Yu , Tikhonov Alexander N
Published: May 2003

The kinetics of the light‐induced redox changes of the photosystem 1 (PS 1) primary donor P700in whole cells of the cyanobacteriaSynechocystissp. PCC 6803 were studied by the electron paramagnetic resonance method. It was shown that the linear photosynthetic electron transport in cyanobacteria was controlled by two main mechanisms: (i) oxygen‐dependent acceleration of electron transfer from PS 1 to NADP+due to activation of the Calvin cycle reactions and (ii) retardation of electron flow between two photosystems governed by a transmembrane proton gradient. In addition to the linear photosynthetic electron transport, cyanobacteria were capable of maintaining alternative pathways involving cyclic electron transfer around PS 1 and respiratory chains.
Molecular mechanisms of insulin receptor substrate protein‐mediated modulation of insulin signalling
Authors: Johnston Anne M. , Pirola Luciano , Van Obberghen Emmanuel
Published: May 2003

The insulin receptor substrate (IRS) proteins act as important mediators of insulin action. Their regulation serves to augment the specificity of the insulin signalling cascade. They can be regulated – both positively and negatively – at the level of phosphorylation, and signalling through these proteins can be further modulated through the actions of SOCS (suppressor of cytokine signalling) proteins. Understanding the mechanisms of IRS regulation will provide further insight into the pathophysiology of insulin resistance and type 2 diabetes.
The ins and outs of leptin receptor activation
Authors: Zabeau Lennart , Lavens Delphine , Peelman Frank , Eyckerman Sven , Vandekerckhove Joël , Tavernier Jan
Published: May 2003

The adipocyte‐derived hormone leptin signals the status of body energy stores by activating its receptor in hypothalamic nuclei. In contrast to the initial expectations, leptin treatment of human obesity was largely unsuccessful. One explanation for this is the marked leptin resistance, which likely operates in part at the receptor level. The leptin receptor is a member of the class I cytokine receptor family, which uses the Janus kinase/signal transducer and activator of transcription pathway as a major signaling route. In this review, we focus on the molecular mechanisms underlying leptin receptor activation. Different modes of leptin‐induced clustering of the ectodomains and the subsequent signaling events will be discussed.
Human tumors associated with Carney complex and germline PRKAR1A mutations: a protein kinase A disease!
Authors: Stergiopoulos Sotirios G. , Stratakis Constantine A.
Published: May 2003

Carney complex (CNC) is a multiple neoplasia syndrome that consists of endocrine (thyroid, pituitary, adrenocortical and gonadal), non‐endocrine (myxomas, nevi and other cutaneous pigmented lesions), and neural (schwannomas) tumors. Primary pigmented nodular adrenocortical disease (PPNAD) is the most common endocrine manifestation of CNC and the only inherited form of Cushing syndrome known to date. In the search of genes responsible for CNC, two chromosomal loci were identified; one (17q22–24) harbored the gene encoding the type I‐α regulatory subunit (RIα) of protein kinase A (PKA), PRKAR1A, a critical component of the cAMP signaling pathway. Here we review CNC and the implications of this discovery for the cAMP and/or PKA's involvement in human tumorigenesis.
A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase
Authors: Stojan Jure , Marcel Véronique , Estrada-Mondaca Sandino , Klaebe Alain , Masson Patrick , Fournier Didier
Published: May 2003

Insect acetylcholinesterase, an enzyme whose catalytic site is located at the bottom of a gorge, can metabolise its substrate in a wide range of concentrations (from 1 μM to 200 mM) since it is activated at low substrate concentrations. It also presents inhibition at high substrate concentrations. Among the various rival kinetic models tested to analyse the kinetic behaviour of the enzyme, the simplest able to explain all the experimental data suggests that there are two sites for substrate molecules on the protein. Binding on the catalytic site located at the bottom of the gorge seems to be irreversible, suggesting that each molecule of substrate which enters the active site gorge is metabolised. Reversible binding at the peripheral site of the free enzyme has high affinity (2 μM), suggesting that this binding increases the probability of the substrate entering the active site gorge. Peripheral site occupation decreases the entrance rate constant of the second substrate molecule to the catalytic site and strongly affects the catalytic activity of the enzyme. On the other hand, catalytic site occupation lowers the affinity of the peripheral site for the substrate (34 mM). These effects between the two sites result both in apparent activation at low substrate concentration and in general inhibition at high substrate concentration.
Negative regulation of interleukin‐1β‐activated neutral sphingomyelinase by protein kinase C in rat mesangial cells
Authors: Kaszkin Marietta , Huwiler Andrea , Scholz Kirsten , van den Bosch Henk , Pfeilschifter Josef
Published: May 2003

Endogenous ceramide is produced by the action of acidic or neutral sphingomyelinases (SMase) in response to stimuli such as proinflammatory cytokines or other inducers of stress. Interleukin‐1β (IL‐1β) is known to stimulate ceramide formation in rat renal mesangial cells; however, the respective subtype of SMase and its regulation have not been investigated. We found that IL‐1β induced an increase in endogenous ceramide levels via the action of a neutral SMase but not an acidic SMase in rat mesangial cells. Cytokine‐induced activation of neutral SMase was inhibited by stimulation of protein kinase C (PKC) by the phorbol ester TPA which caused a reduction of ceramide back to control levels. This inhibitory effect of TPA was reversed by the specific PKC‐inhibitor Ro‐318220. Long‐term incubation (24 h) of mesangial cells with TPA, which downregulates PKC‐α, ‐δ, and ‐ϵ isoenzymes, resulted in a recovery of IL‐1β‐stimulated neutral SMase activity as well as ceramide formation. These data implicate an important modulatory function of PKC in ceramide production in IL‐1β‐activated mesangial cells.
Bacteriophage lambda surface display of a bacterial biotin acceptor domain reveals the minimal peptide size required for biotinylation
Authors: Stolz Jürgen , Ludwig Andreas , Sauer Norbert
Published: May 2003

Phage display is a powerful technique for identifying specific ligands to a given target. In this work random peptides derived from the biotin accepting domain of the Klebsiella pneumoniae oxaloacetate decarboxylase were displayed on bacteriophage lambda heads to determine the minimal sequence length that is necessary to effect biotinylation in vivo. Phages with a functional biotinylation domain were identified after affinity purification with immobilised avidin. All biotinylated phages isolated this way were found to have a sequence of 66 amino acids from the parental protein in common. This minimal biotinylation domain is fully functional as a biotin acceptor and more resistant to proteolytic attack compared to domains of larger size derived from the same protein. The data present the first example of a posttranslational protein modification analysed in a phage display system. Moreover, a biotin domain of reduced size and improved stability was identified, that should be superior to the larger parental protein as a tag to generate biotinylated fusion proteins.
The effects of the Rho‐kinase inhibitor Y‐27632 on arachidonic acid‐, GTPγS‐, and phorbol ester‐induced Ca2+‐sensitization of smooth muscle
Authors: Fu Xiaohong , Gong Ming Cui , Jia Taiping , Somlyo Avril V , Somlyo Andrew P
Published: May 2003

The effects of the Rho‐kinase inhibitor, Y‐27632 [1] on Ca2+‐sensitization of force induced by arachidonic acid (AA), phorbol 12,13‐dibutyrate (PDBu), GTPγS, and by the stable thromboxane analog, 9,11‐dideoxy‐9α,11α‐methanoepoxy‐PGF2α (U‐46619), were determined in α‐toxin‐permeabilized smooth muscles. Y‐27632 relaxed (up to 99%) Ca2+‐sensitization by GTPγS (10 μM) and U‐46619 (1 μM), but not by PDBu (20 μM), and reduced GTPγS‐induced myosin light chain (MLC20) phosphorylation from 28% to 17% (P=0.002). GTPγS‐induced force sensitization was inhibited by Y‐27632 more potently when the inhibitor was added during the plateau of force than prior to stimulation. In α‐toxin‐permeabilized smooth muscle, Y‐27632 inhibited AA (50 μM)‐induced Ca2+‐sensitization of force (by 66±1.3%) and reduced MLC20 phosphorylation. In contrast, Y‐27632 did not relax force Ca2+‐sensitized by AA in smooth muscle permeabilized with Triton X‐100. We conclude that (i) AA induces Ca2+‐sensitization through dual mechanisms, one mediated by Rho‐kinase (or a related kinase), and (ii) Rho‐kinase is not required for phorbol ester‐induced Ca2+‐sensitization.
Inhibition of skeletal muscle sarcoplasmic reticulum Ca2+‐ATPase by nitric oxide
Authors: Ishii Toshiaki , Sunami Osamu , Saitoh Noriko , Nishio Hideaki , Takeuchi Tadayoshi , Hata Fumiaki
Published: May 2003

The effects of nitric oxide on the activities of thapsigargin‐sensitive sarcoplasmic reticulum Ca2+‐ATPase (SERCA) and Ca2+ uptake by sarcoplasmic reticulum (SR) membranes prepared from white skeletal muscle of rabbit femoral muscle were studied. Pretreatment of the SR preparations with nitric oxide at concentrations of up to 250 μM for 1 min decreased the SERCA activity concentration dependently, and also decreased their Ca2+ uptake. Both these effects of nitric oxide were reversible. Inhibitors of guanylyl cyclase and protein kinase G (PKG) had no significant effect on the nitric oxide‐induced inhibitions of SERCA and Ca2+ uptake. Moreover, dithiothreitol did not reverse the inhibitory effects of nitric oxide on SERCA and Ca2+ uptake. These findings suggest that nitric oxide inhibits SERCA, mainly SERCA 1, of rabbit femoral skeletal muscle by an action independent of the cyclic GMP‐PKG system or oxidation of thiols, and probably by a direct action on SERCA protein.
Early pleistocene lake deposits and Lower Paleolithic finds in Nahal (wadi) Zihor, Southern Negev desert, Israel
Authors: Ginat Hanan , Zilberman Ezra , Saragusti Idit
Published: May 2003

An Early Pleistocene fluvio-lacustrine sequence from the extremely arid southern Negev desert, Israel, indicates that climatic conditions during that period were humid enough to support the formation of a lake (Lake Zihor) there. The lacustrine sediments were deposited in a tectonic valley that developed along the Zihor tectonic line after the deposition of the Pliocene Arava Formation. They intercalate with fluvial sediments that contain several horizons of reddish calcic paleosols. The lacustrine sequence comprises three sedimentary cycles. Each starts with a dark clay layer overlain by a white limestone unit, both of which contain abundant freshwater fauna, and terminates with a green detrital limestone that contains only one type of brackish-water ostracod. The top of each green limestone unit is characterized by pedogenic features and microrelief, indicating desiccation of the lake and soil development. Isotope data support the hypothesis that the white limestone was deposited in an open, freshwater lake. The incision of the present channel of Nahal (wadi) Zihor in the lacustrine sediments is manifested by a series of rock-cut and fluvial terraces (Q1–Q4) capped by gypsic-salic soils, which reflect the onset of the present, extremely arid climate. Over 100 find-spots and larger occurrences of prehistoric artifacts assigned to the Lower Paleolithic were discovered near Lake Zihor. On the basis of techno-typological and stratigraphic considerations, these assemblages are divided into two groups, the first of which may be contemporaneous with the lake, while the second is found mainly on the younger Q1 and Q2 terraces. It is estimated that the lake existed for more than 100,000 years. The climate during this period was probably semiarid, but water budget calculations suggest that, in addition to surface runoff, seepage of groundwater along the Zihor Line contributed a considerable amount of water to the lake.