bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterization and functional expression of dihydroxypterocarpan 6a‐hydroxylase, an enzyme specific for pterocarpanoid phytoalexin biosynthesis in soybean (Glycine max L.)
Authors: Schopfer Christel R , Kochs Georg , Lottspeich Friedrich , Ebel Jürgen
Published: July 2002

Four cytochrome P450‐dependent enzymes, among them dihydroxypterocarpan 6a‐hydroxylase (D6aH), are specifically involved in the elicitor‐inducible biosynthesis of glyceollins, the phytoalexins of soybean. Here we report that CYP93A1 cDNA, which we isolated previously from elicitor‐induced soybean cells, codes for a protein with D6aH activity. Analysis of the catalytic properties of recombinant CYP93A1 expressed in yeast, its NADPH dependency, stereoselectivity and high substrate affinity confirmed that D6aH is the physiological function of CYP93A1. It thus represents the first isoflavonoid‐specific CYP to be characterized at the molecular level. In elicitor‐treated soybean cells producing phytoalexins, increases in D6aH activity were correlated with elevated transcript levels which indicates that expression of the enzyme is regulated at the level of transcription. Therefore, CYP93A1 cDNA can be used as a specific molecular marker for the inducible defense response against pathogen attack.
Direct association of presenilin‐1 with β‐catenin
Authors: Murayama Miyuki , Tanaka Shoji , Palacino James , Murayama Ohoshi , Honda Toshiyuki , Sun Xiaoyan , Yasutake Kaori , Nihonmatsu Naomi , Wolozin Benjamin , Takashima Akihiko
Published: July 2002

Families bearing mutations in the presenilin‐1 (PS1) gene develop Alzheimer's disease (AD). However, the mechanism through which PS1 causes AD is unclear. The co‐immunoprecipitation with PS1 in transfected COS‐7 cells indicates that PS1 directly interacts with endogenous β‐catenin, and the interaction requires residues 322–450 of PS1 and 445–676 of β‐catenin. Both proteins are co‐localized in the endoplasmic reticulum. Over‐expression of PS1 reduces the level of cytoplasmic β‐catenin, and inhibits β‐catenin‐T cell factor‐regulated transcription. These results indicate that PS1 plays a role as inhibitor of the β‐catenin signal, which may be connected with the AD dysfunction.
Multiple crystal forms of hexokinase I: new insights regarding conformational dynamics, subunit interactions, and membrane association
Authors: Aleshin Alexander E , Fromm Herbert J , Honzatko Richard B
Published: July 2002

Hexokinase I is comprised of homologous N‐ and C‐terminal domains, and binds to the outer membrane of mitochondria. Reported here is the structure of a new crystal form of recombinant human hexokinase I, which complements existing crystal structures. Evidently, in some packing environments and even in the presence of glucose and glucose 6‐phosphate the N‐terminal domain (but not the C‐terminal domain) can undergo oscillations between closed and partially opened conformations. Subunit interfaces, present in all known crystal forms of hexokinase I, promote the formation of linear chains of hexokinase I dimers. Presented is a model for membrane‐associated hexokinase I, in which linear chains of hexokinase I dimers are stabilized by interactions with mitochondrial porin.
pH‐sensitive liposomes for receptor‐mediated delivery to chicken hepatoma (LMH) cells
Authors: Škalko Nataša , Peschka Regine , Altenschmidt Uwe , Lung Andreas , Schubert Rolf
Published: July 2002

pH‐sensitive liposomes composed of dioleoylphosphatidylethanolamine and cholesterol hemisuccinate (3:2 mol/mol) bearing the N‐acetylglucosamine derivative of bovine serum albumin (N‐Ac‐BSA) were applied for receptor‐mediated delivery in chicken hepatoma (LMH) cells expressing the N‐Ac‐BSA‐binding asialoglycoprotein receptor. Fluorescently labeled dextran was entrapped in liposomes by a modified freeze‐thawing method (encapsulation efficiency of 23%). A novel method of coupling proteins onto the surface of preformed liposomes yielded a coupling efficiency of 60–70%. The association of pH‐sensitive and lecithin liposomes with LMH cells was monitored by fluorescence‐activated cell sorting and confocal microscopy. Prerequisites for receptor‐mediated delivery to LMH cells were both the pH sensitivity of liposomes and the presence of N‐Ac‐BSA on the liposomal surface.
Characterization of early induced genes in Arabidopsis thaliana responding to bacterial inoculation: identification of centrin and of a novel protein with two regions related to kinase domains
Authors: Cordeiro M.C.R. , Piqueras R. , de Oliveira D.E. , Castresana C.
Published: July 2002

The early response to bacterial inoculation has been investigated and two Arabidopsis genes, ap3.3a and ap4.3a have been characterized. The AP3.3A protein showed high identity to centrin, a ubiquitous cytoskeletal protein first identified in unicellular green alga. Amino‐acid sequence analyses of the AP4.3A protein indicates that the second gene characterized encodes an unusual protein with two putative kinase domains. Expression of ap3.3a and ap4.3a was rapidly induced after pathogen inoculation. A role of ap3.3a in plant defense could be postulated based on its preferential induction during the incompatible interactions analyzed. In contrast, activation of ap4.3a was not specific and could be related to a more general stress response.
cDNA cloning of Brassica napus malonyl‐CoA:ACP transacylase (MCAT) (fab D) and complementation of an E. coli MCAT mutant
Authors: Simon J.W. , Slabas A.R.
Published: July 2002

The GenBank database was searched using the E. coli malonyl CoA:ACP transacylase (MCAT) sequence, for plant protein/cDNA sequences corresponding to MCAT, a component of plant fatty acid synthetase (FAS), for which the plant cDNA has not been isolated. A 272‐bp Zea mays EST sequence (GenBank accession number: AA030706) was identified which has strong homology to the E. coli MCAT. A PCR derived cDNA probe from Zea mays was used to screen a Brassica napus (rape) cDNA library. This resulted in the isolation of a 1200‐bp cDNA clone which encodes an open reading frame corresponding to a protein of 351 amino acids. The protein shows 47% homology to the E. coli MCAT amino acid sequence in the coding region for the mature protein. Expression of a plasmid (pMCATrap2) containing the plant cDNA sequence in Fab D89, an E. coli mutant, in MCAT activity restores growth demonstrating functional complementation and direct function of the cloned cDNA. This is the first functional evidence supporting the identification of a plant cDNA for MCAT.
A second isoform of gonadotropin‐releasing hormone is present in the brain of human and rodents
Authors: Chen Alon , Yahalom Dror , Ben-Aroya Nurit , Kaganovsky Ella , Okon Eli , Koch Yitzhak
Published: July 2002

Gonadotropin‐releasing hormone‐I (GnRH‐I), present in the mammalian hypothalamus, regulates reproduction. In this study we demonstrate, for the first time, that an additional isoform of GnRH, [His5, Trp7, Tyr8] GnRH‐I (GnRH‐II) is present in the brain of the mouse, rat and human. Human and rat brain extracts contain two isoforms of GnRH, GnRH‐I and GnRH‐II, which exhibited identical chromatographic properties to the respective synthetic peptides, in high performance liquid chromatography. Using immunohistochemical techniques we have found that GnRH‐II is present in neuronal cells that are localized mainly in the periaqueductal area as well as in the oculomotor and red nuclei of the midbrain. It is of interest to note that in the hypogonadal mouse, although the GnRH‐I gene is deleted, GnRH‐II is present. Substantial concentrations of GnRH‐II are also present in the hypothalamus and stored in the human pituitary stalk or in the mouse median eminence. By using reverse transcription (RT)‐PCR we have also found that while GnRH‐II is not expressed in the cerebellum, it is expressed in all three structures of the brain stem: midbrain, pons and medulla oblongata.
Okadaic acid‐induced apoptosis of HL60 leukemia cells is preceded by destabilization of bcl‐2 mRNA and downregulation of bcl‐2 protein
Authors: Riordan Fiona A. , Foroni Letizia , Hoffbrand A.Victor , Mehta Atul B. , Wickremasinghe R.Gitendra
Published: July 2002

We have studied the actions of the protein phosphatase inhibitor okadaic acid (OA) on the expression of bcl‐2 in HL60 human leukemia cells. OA induced downregulation of bcl‐2 mRNA and protein prior to the induction of apoptosis. Downregulation of bcl‐2 mRNA levels did not result from actions of OA on the bcl‐2 upstream negative response element. Nuclear run‐off analyses confirmed that OA did not affect bcl‐2 gene transcription. However, OA caused a rapid increase in the rate of degradation of bcl‐2 mRNA. Therefore, OA induces downregulation of bcl‐2 expression via destabilization of its transcript. The constitutive action of an OA‐sensitive protein phosphatase may therefore maintain HL60 cell survival by blocking bcl‐2 mRNA degradation.
Cloning, expression, and localization of a novel γ‐adaptin‐like molecule
Authors: Lewin David A. , Sheff David , Ooi Chean Eng , Whitney J.Andrew , Yamamoto Ellen , Chicione Linda M. , Webster Paul , Bonifacino Juan S. , Mellman Ira
Published: July 2002

We describe the cloning, expression, and localization of γ2‐adaptin, a novel isoform of γ‐adaptin. The predicted human and mouse γ2‐adaptin proteins are ∼90 kDa and 64.4% and 61.7% identical to γ‐adaptin, respectively. γ2‐Adaptin was localized to the Golgi, its localization distinct from γ‐adaptin. The membrane association of γ‐ and γ2‐adaptin could further be distinguished by differential sensitivity to the fungal metabolite brefeldin A, γ2‐adaptin binding being insensitive to drug treatment. Together, these results suggest that γ2‐adaptin plays a role in membrane transport distinct from that played by γ‐adaptin.
Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver β‐glucosidase activity
Authors: Day Andrea J , DuPont M.Susan , Ridley Saxon , Rhodes Mike , Rhodes Michael J.C , Morgan Michael R.A , Williamson Gary
Published: July 2002

Flavonoid and isoflavonoid glycosides are common dietary phenolics which may be absorbed from the small intestine of humans. The ability of cell‐free extracts from human small intestine and liver to deglycosylate various (iso)flavonoid glycosides was investigated. Quercetin 4′‐glucoside, naringenin 7‐glucoside, apigenin 7‐glucoside, genistein 7‐glucoside and daidzein 7‐glucoside were rapidly deglycosylated by both tissue extracts, whereas quercetin 3,4′‐diglucoside, quercetin 3‐glucoside, kaempferol 3‐glucoside, quercetin 3‐rhamnoglucoside and naringenin 7‐rhamnoglucoside remained unchanged. The K
m for hydrolysis of quercetin 4′‐glucoside and genistein 7‐glucoside was ∼32±12 and ∼14±3 μM in both tissues respectively. The enzymatic activity of the cell‐free extracts exhibits similar properties to the cytosolic broad‐specificity β‐glucosidase previously described in mammals.
Spermine causes caspase activation in leukaemia cells
Authors: Stefanelli Claudio , Bonavita Francesca , Stanic' Ivana , Mignani Monica , Facchini Annalisa , Pignatti Carla , Flamigni Flavio , Caldarera Claudio M
Published: July 2002

Exposure of several leukaemia cell types to the polyamine spermine triggered caspase activation. In HL60 cells, the onset of caspase activity correlated with the accumulation of spermine, and was accompanied by the processing of the caspase‐3 precursor and the digestion of the substrate proteins PARP and gelsolin. Spermine also induced the accumulation of cytochrome c in the cytosol. Caspase activation triggered by spermine was not blocked by antioxidants or inhibition of polyamine oxidase. The deregulation of polyamine uptake strongly sensitised the cells to spermine‐induced caspase activation. These data show that an excessive intracellular level of spermine triggers caspase activation that is not mediated by oxidative mechanisms, and suggest a model where elevated free cytosolic polyamines may act as transducers of a death message.
Factors responsible for inhibiting the motility of zoospores of the phytopathogenic fungus Aphanomycescochlioides isolated from the non‐host plant Portulacaoleracea
Authors: Mizutani Masanori , Hashidoko Yasuyuki , Tahara Satoshi
Published: July 2002

In a survey of plant secondary metabolites regulating the behaviour of Aphanomyces cochlioides zoospores, we found that root extracts of Portulaca oleracea inhibited zoospore motility. Bioassay‐directed fractionation of Portulaca constituents revealed that the inhibitory activity was dependent on the interaction of two chemically different factors. These were identified as a phenolic compound, N‐trans‐feruloyltyramine, which by itself was active as a zoospore stimulant, and an acidic compound, 1‐linoleoyl‐2‐lysophosphatidic acid monomethyl ester, which had zoospore‐repellent activity. When Chromosorb W AW particles coated with a mixture of these pure compounds were bioassayed in Petri dishes, the inhibitory effect on zoospore motility was identical with that caused by root tip or root extracts of P. oleracea. Inhibited zoospores rapidly settled to the bottom of the Petri dishes where they initially encysted, and then germinated within 1–2 h. This is the first report of factors which inhibit zoospore motility without killing or bursting the zoospores.
Complex formation of the nonstructural protein 3 of hepatitis C virus with the p53 tumor suppressor
Authors: Ishido Satoshi , Hotta Hak
Published: July 2002

By co‐immunoprecipitation analysis, we demonstrated that wt‐p53 formed a complex with non‐structural protein (NS) 3 of hepatitis C virus, both in the absence and the presence of NS4A, a viral cofactor that strongly associates with NS3. Deletional analysis revealed that a portion near the N‐terminus of NS3 (amino acids (aa) 1055 and 1200), which is different from the NS4A binding site, was necessary for the complex formation with wt‐p53. On the other hand, a portion near the C‐terminus of wt‐p53 (aa 301–360), which has been reported to contain the oligomerization domain, was important for the complex formation with NS3.
Synthesis and structure‐function study about tenecin 1, an antibacterial protein from larvae of Tenebrio molitor
Authors: Lee Keun Hyeung , Hong Sung Yu , Oh Jong Eun
Published: July 2002

Tenecin 1, an inducible antibacterial protein secreted in the larvae of Tenebrio molitor, has a long N‐terminal loop and common structural feature of insect defensin family corresponding to cysteine stabilized α/β motif. To study the function of the N‐terminal loop and disulfide bridges, N‐terminal loop deleted tenecin 1, reduced tenecin 1 and tenecin 1 were chemically synthesized and their activities were measured. N‐terminal loop deleted tenecin and reduced tenecin 1 did not show antibacterial activity. Circular dichroism (CD) spectroscopy data revealed that the α‐helical content of tenecin 1 and the other proteins increased in the presence of 50% (v/v) trifluoroethanol (TFE) and the α‐helical content of tenecin 1 was much higher than that of the other proteins in buffer with or without 50% (v/v) TFE. These results suggest that disulfide bridges are necessary for the activity structure and the N‐terminal loop plays an important role in the increase of α‐helix in the membrane mimetic environment and the activity.
Cooperative cyclic interactions involved in metal binding to pairs of sites in EF‐hand proteins
Authors: Biekofsky Rodolfo R , Feeney James
Published: July 2002

This study focuses on a closed net of electron‐pair donor–acceptor interactions, present in the core of all metal‐bound EF‐hand pairs, that link both metal ions across a short two‐stranded β‐sheet. A molecular model based on the above cycle of interactions was studied using semi‐empirical molecular orbital quantum mechanical methods. The calculations indicate that the interactions in the model cycle are cooperative, that is, that the interaction energy of the cyclic structure is greater than that of the sum of isolated interactions between its components. The cooperativity in this cycle can be attributed to an increase in the stability of the interactions resulting from a mutual polarisation of the associated groups. The predicted polarisation of the amide groups in the cycle is in agreement with experimental NMR 15N deshielding observed for these amide groups upon metal binding. Experimental observations of strengthening of the β‐sheet hydrogen bonds are also consistent with the model calculations. By this mechanism, the binding of the first metal ion would enhance the binding of the second metal ion, and thus, the intradomain cooperativity in cation binding of calmodulin and related EF‐hand proteins can be ascribed, at least partly, to this short‐range molecular mechanism.
Modulation of the UVA activation of haem oxygenase, collagenase and cyclooxygenase gene expression by epigallocatechin in human skin cells
Authors: Soriani Marco , Rice-Evans Catherine , Tyrrell Rex M
Published: July 2002

We have investigated the modifying effects of epigallocatechin, a major polyphenolic constituent of green tea, on ultraviolet‐A‐activated gene expression in human fibroblasts and keratinocytes using the stress responsive enzymes: haem oxygenase‐1, interstitial collagenase and cyclooxygenase‐2. Although epigallocatechin strongly reduced ultraviolet‐A‐induced haem oxygenase‐1 activation in skin‐derived fibroblasts, the same compound activated collagenase and cyclooxygenase expression. In a keratinocyte cell line, ultraviolet‐A‐mediated haem oxygenase‐1 over‐expression was low and epigallocatechin failed to modulate it further. In contrast to the results with fibroblasts, ultraviolet‐A activation of cyclooxygenase in keratinocytes was reduced by epigallocatechin. The results indicate that the effect of this green tea polyphenol on cellular stress responses is complex and may involve direct effects on signal transduction as well as changes that may be associated with its antioxidant activity.
Sulfur K‐edge X‐ray absorption spectroscopy for determining the chemical speciation of sulfur in biological systems
Authors: Pickering Ingrid J. , Prince Roger C. , Divers Thomas , George Graham N.
Published: July 2002

Sulfur is an essential biological element, yet its biochemistry is only partially understood because there are so few tools for studying this element in biological systems. X‐ray absorption spectroscopy provides a unique approach to determining the chemical speciation of sulfur in intact biological samples. Different biologically relevant sulfur compounds show distinctly different sulfur K‐edge X‐ray absorption spectra, and we show here, as an example, that this allows the deconvolution of the sulfur species in equine blood.
Transcellular regulation of cell respiration by nitric oxide generated by activated macrophages
Authors: Brown Guy C , Foxwell Neale , Moncada Salvador
Published: July 2002

A macrophage cell line (J774), activated with interferon‐γ and endotoxin to express the inducible form of NO synthase (iNOS), immediately inhibited the cellular respiration of co‐incubated L‐929 fibroblasts or non‐activated J774 macrophages. The inhibition was potent, rapid and reversible when the NO was removed by adding oxyhaemoglobin or by inhibiting iNOS. Exogenously added NO also rapidly and reversibly inhibited cellular respiration over the same range of NO concentrations. This inhibition was competitive with oxygen and due to direct inhibition of cytochrome oxidase. Thus, NO generated by one cell can regulate the respiration of adjacent cells, supporting the hypothesis that NO may be a physiological and/or pathological regulator of cellular respiration, via its inhibition of cytochrome oxidase.
Negative charge at the protein kinase CK2 site enhances recognition of the SV40 large T‐antigen NLS by importin: effect of conformation
Authors: Xiao Chong-Yun , Jans Patricia , Jans David A.
Published: July 2002

SV40 large tumor‐antigen (T‐ag) nuclear import is enhanced by the protein kinase CK2 (CK2) site (Ser111Ser112) flanking the nuclear localization sequence (NLS). Here we use site‐directed mutagenesis to examine the influence of negative charge and conformation at the site on T‐ag nuclear import and recognition by the NLS‐binding importin subunits. Negative charge through aspartic acid in place of Ser111 simulated CK2 phosphorylation in enhancing nuclear accumulation to levels well above those of proteins lacking a functional CK2 site. This was shown to be through enhancement of T‐ag NLS recognition by importin using an ELISA‐based assay. Asp112‐substituted mutants containing proline at positions 109, 110 (wild‐type position) or 111 were compared to assess the role of conformation at the CK2 site. Maximal nuclear import of the protein with Pro109 was lower than that of the Pro110 derivative, with the Pro111 variant even lower, these differences also being attributable to effects on importin binding. All results indicate a correlation of the initial nuclear import rate with the importin binding affinity, demonstrating that NLS recognition by importin is a key rate‐determining step in nuclear import.
Eukaryotic mRNAs encoding abundant and scarce proteins are statistically dissimilar in many structural features
Authors: Kochetov Alex V , Ischenko Igor V , Vorobiev Denis G , Kel Alexander E , Babenko Vladimir N , Kisselev Lev L , Kolchanov Nikolay A
Published: July 2002

It is well known that non‐coding mRNA sequences are dissimilar in many structural features. For individual mRNAs correlations were found for some of these features and their translational efficiency. However, no systematic statistical analysis was undertaken to relate protein abundance and structural characteristics of mRNA encoding the given protein. We have demonstrated that structural and contextual features of eukaryotic mRNAs encoding high‐ and low‐abundant proteins differ in the 5′ untranslated regions (UTR). Statistically, 5′ UTRs of low‐expression mRNAs are longer, their guanine plus cytosine content is higher, they have a less optimal context of the translation initiation codons of the main open reading frames and contain more frequently upstream AUG than 5′ UTRs of high‐expression mRNAs. Apart from the differences in 5′ UTRs, high‐expression mRNAs contain stronger termination signals. Structural features of low‐ and high‐expression mRNAs are likely to contribute to the yield of their protein products.
13C isotopomer analysis of glutamate by heteronuclear multiple quantum coherence‐total correlation spectroscopy (HMQC‐TOCSY)
Authors: Carvalho Rui A. , Jeffrey F.Mark H. , Sherry A.Dean , Malloy Craig R.
Published: July 2002

13C has become an important tracer isotope for studies of intermediary metabolism. Information about relative flux through pathways is encoded by the distribution of 13C isotopomers in an intermediate pool such as glutamate. This information is commonly decoded either by mass spectrometry or by measuring relative multiplet areas in a 13C NMR spectrum. We demonstrate here that groups of glutamate 13C isotopomers may be quantified by indirect detection of protons in a 2D HMQC‐TOCSY NMR spectrum and that fitting of these data to a metabolic model provides an identical measure of the 13C fractional enrichment of acetyl‐CoA and relative anaplerotic flux to that given by direct 13C NMR analysis. The sensitivity gain provided by HMQC‐TOCSY spectroscopy will allow an extension of 13C isotopomer analysis to tissue samples not amenable to direct 13C detection (∼10 mg soleus muscle) and to tissue metabolites other than glutamate that are typically present at lower concentrations.
Changes in cytoplasmic ATP concentration parallels changes in ATP‐regulated K+‐channel activity in insulin‐secreting cells
Authors: Köhler Martin , Norgren Svante , Berggren Per-Olof , Fredholm Bertil B , Larsson Olof , Rhodes Christopher J , Herbert Terence P , Luthman Holger
Published: July 2002

Changes in cytoplasmic ATP concentration were monitored in intact insulin‐producing cells and correlated to changes in the activity of ATP‐sensitive K+‐channels (KATPchannels). Luciferase was introduced into HIT M2.2 cells and whole pancreatic islets by transient expression of firefly (Photinus pyralis) luciferase cDNA. In transfected cells, extracellular addition of luciferin increased the luminescence signal to a maximum within 50–120 s. Addition of 1 μM of the mitochondrial uncoupler FCCP decreased the luminescence, an effect partly reversed upon withdrawal of the compound. High concentrations of glucose increased cytoplasmic free ATP concentration. Changes in the luminescence signal were accompanied by changes in activity of the ATP‐sensitive K+‐channel. Transfection per se did not deteroriate cell function, as verified by experiments showing similar changes in cytoplasmic free Ca2+‐concentration, [Ca2+]i, in both transfected and non‐transfected cells. By measuring the cytoplasmic ATP concentration and KATPchannel activity under similar experimental conditions, it was possible to establish, for the first time, a direct relationship between these two parameters. This indeed suggests that the cytoplasmic ATP concentration has a crucial role in the regulation of KATPchannel activity under physiological conditions.
9‐cis Retinoic acid induces the expression of the uncoupling protein‐2 gene in brown adipocytes
Authors: Carmona M.C , Valmaseda A , Iglesias R , Mampel T , Viñas O , Giralt M , Villarroya F
Published: July 2002

The expression of uncoupling protein‐2 (UCP2) mRNA is up‐regulated during the differentiation of brown adipocytes in primary culture. When differentiation of brown adipocytes is impaired, UCP2 mRNA expression is down‐regulated. 9‐cis Retinoic acid causes a dose‐dependent induction of UCP2 mRNA levels in brown adipocytes, whereas all‐trans retinoic acid has no effect. Specific agonists of retinoid X receptors (RXR) induce UCP2 mRNA expression, whereas specific activators of retinoic acid receptors do not. 9‐cis Retinoic acid, acting through RXR receptors, is identified as a major regulator of the expression of the UCP2 gene in the brown fat cell.
Isolation of p‐hydroxycinnamaldehyde as an antibacterial substance from the saw fly, Acantholyda parki S
Authors: Leem Jae Yoon , Jeong Im Ju , Park Kyu Tek , Park Ho Yong
Published: July 2002

We purified an antibacterial substance from larvae of the saw fly, Acantholyda parki S., and identified its molecular structure as p‐hydroxycinnamaldehyde. We then synthesized it by reduction of p‐hydroxycinnamic acid. The antibacterial activity of the synthetic p‐hydroxycinnamaldehyde was equal to that of the authentic substance. This molecule was found to have a broad antibacterial spectrum against not only Gram‐negative, but also Gram‐positive bacteria. Furthermore, it showed antifungal activity against Candida albicans. We suggest that this substance may play a role in the defense system of this insect. This is the first report of p‐hydroxycinnamaldehyde of animal origin.
Structure to 1.9 Å resolution of a complex with herpes simplex virus type‐1 thymidine kinase of a novel, non‐substrate inhibitor: X‐ray crystallographic comparison with binding of aciclovir
Authors: Bennett Matthew S , Wien Frank , Champness John N , Batuwangala Thilina , Rutherford Thomas , Summers William C , Sun Hongmao , Wright George , Sanderson Mark R
Published: July 2002

Treatment of herpes infections with nucleoside analogues requires as an initial step the activation of the compounds by thymidine kinase. As an aid to developing more effective chemotherapy, both for treatment of recurrent herpes infection and in gene therapy systems where thymidine kinase is expressed, two high‐resolution X‐ray structures of thymidine kinase have been compared: one with the relatively poor substrate aciclovir (Zovirax), the other with a synthetic inhibitor having an N
2‐substituted guanine. Both compounds have similar binding modes in spite of their size difference and apparently distinct ligand properties.
Interferon‐γ inhibits the myofibroblastic phenotype of rat palatal fibroblasts induced by transforming growth factor‐β1 in vitro
Authors: Yokozeki Masahiko , Baba Yoshiyuki , Shimokawa Hitoyata , Moriyama Keiji , Kuroda Takayuki
Published: July 2002

Interferon‐γ (IFN‐γ), a multifunctional cytokine, has been noted as a potential therapeutic agent for various fibrotic disorders, including excessive scar tissue formation. We previously reported that transforming growth factor‐β1 (TGF‐β1) induced the myofibroblastic phenotype in palatal fibroblasts derived from palatal mucosa, and that such effects might have a close link to palatal scar formation. In the present study, we examined the effects of IFN‐γ on TGF‐β1‐pretreated palatal fibroblasts for the purpose of clarifying the suppressive potency against myofibroblastic phenotype expression in vitro. IFN‐γ significantly altered the spindle morphology of TGF‐β1‐pretreated palatal fibroblasts into the polygonal one that was similar to the non‐treated palatal fibroblasts. This change was parallel with a decrease in the expression of α‐smooth muscle actin protein, a marker for myofibroblast, as determined by immunoblot analysis. Northern blot analysis showed that IFN‐γ inhibited proα2(I) collagen mRNA expression that was stimulated by TGF‐β1 pretreatment for 24 h. Furthermore, IFN‐γ decreased the cell contractility enhanced by TGF‐β1 pretreatment for 24 h in a three‐dimensional collagen gel culture system. These results suggest that IFN‐γ may have negative effects with regard to controlling the myofibroblastic phenotype induced by TGF‐β1 in palatal fibroblasts.
A recombinant single‐chain antibody fragment that neutralizes toxin II from the venom of the scorpion Androctonus australis hector
Authors: Mousli Mohamed , Devaux Christiane , Rochat Hervé , Goyffon Max , Billiald Philippe
Published: July 2002

Monoclonal antibody 4C1 specifically binds to and neutralizes the most potent neurotoxin (AahII) of the scorpion Androctonus australis. The cDNAs encoding the variable regions of this antibody were isolated by PCR‐mediated cloning. A single‐chain Fv gene was engineered and expressed in Escherichia coli. The recombinant protein had neutralizing activity similar to that of the intact antibody in vitro and in vivo. We have thus neutralized the pharmacological and biological properties of a scorpion neurotoxin with a single‐chain Fv, which opens new perspectives for the treatment of envenomizations.
Expression of functional Na+/H+ antiporters of Helicobacter pylori in antiporter‐deficient Echerichia coli mutants
Authors: Inoue Hiroki , Sakurai Tatsuya , Ujike Satoshi , Tsuchiya Tomofusa , Murakami Hiroshi , Kanazawa Hiroshi
Published: July 2002

An open reading frame with a sequence homologous to Escherichia coli Na+/H+ antiporter A (ENhaA) was found in the total genomic sequence of Helicobacter pylori, a pathogenic bacterium of gastric inflammation, and was named HNhaA. The primary sequences and the hydropathy profiles of ENhaA and HNhaA were very homologous except for one additional region found in HNhaA. This sequence has about 40 hydrophilic amino acid residues inserted at the position next to residue 235 of ENhaA which corresponds to residue 245 of HNhaA. HNhaA was expressed in E. coli mutants deficient in Na+/H+ antiporters and complemented the salt‐sensitive phenotype of the mutants. Membrane vesicles prepared from these transformants of HNhaA using mutants deficient in the antiporters had the antiporter activities. Surprisingly, the antiporter activity in the transformant membranes was high at acidic and neutral pH, while ENhaA did not function at these pHs. A hydrophilic region around residue 235 in ENhaA and the additional hydrophilic region of about 40 residues in the same region found in HNhaA might be responsible for this difference in activity by acting as putative pH sensors.
The Saccharomyces cerevisiae succinate‐ubiquinone reductase contains a stoichiometric amount of cytochrome b562
Authors: Oyedotun Kayode S , Lemire Bernard D
Published: July 2002

The Saccharomyces cerevisiae succinate‐ubiquinone reductase or succinate dehydrogenase (SDH) is a tetramer of non‐equivalent subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. In most organisms, SDH contains one or two endogenous b‐type hemes. However, it is widely believed that the yeast SDH does not contain heme. In this report, we demonstrate the presence of a stoichiometric amount of cytochrome b
562 in the yeast SDH. The cytochrome is detected as a peak present in fumarate‐oxidized, dithionite‐reduced mitochondria. The peak is centered at 562 nm and is present at a heme:covalent FAD molar ratio of 0.92±0.11. The cytochrome is not detectable in mitochondria isolated from SDH3 and SDH4 deletion strains. These observations strongly support our conclusion that cytochrome b
562 is a component of the yeast SDH.
The structure of human parathyroid hormone‐related protein(1–34) in near‐physiological solution
Authors: Weidler Marcus , Marx Ute C , Seidel Gabi , Schäfer Wolfgang , Hoffmann Eike , Eßwein Angelika , Rösch Paul
Published: July 2002

Parathyroid hormone‐related protein plays a major role in the pathogenesis of humoral hypercalcemia of malignancy. Under normal physiological conditions, parathyroid hormone‐related protein is produced in a wide variety of tissues and acts in an autocrine or paracrine fashion. Parathyroid hormone‐related protein and parathyroid hormone bind to and activate the same G‐protein‐coupled receptor. Here we present the structure of the biologically active NH2‐terminal domain of human parathyroid hormone‐related protein(1–34) in near‐physiological solution in the absence of crowding reagents as determined by two‐dimensional proton magnetic resonance spectroscopy. An improved strategy for structure calculation revealed the presence of two helices, His‐5–Leu‐8 and Gln‐16–Leu‐27, connected by a flexible linker. The parathyroid hormone‐related protein(1–34) structure and the structure of human parathyroid hormone(1–37) as well as human parathyroid hormone(1–34) are highly similar, except for the well defined turn, His‐14–Ser‐17, present in parathyroid hormone. Thus, the similarity of the binding affinities of parathyroid hormone and parathyroid hormone‐related protein to their common receptor may be based on their structural similarity.
Activation of phospholipase C‐Ca2+ system by sphingosine 1‐phosphate in CHO cells transfected with Edg‐3, a putative lipid receptor
Authors: Sato Koichi , Kon Junko , Tomura Hideaki , Osada Mizuho , Murata Naoya , Kuwabara Atsushi , Watanabe Tomoko , Ohta Hideo , Ui Michio , Okajima Fumikazu
Published: July 2002

Sphingosine 1‐phosphate (S1P) induces phospholipase C (PLC) activation and Ca2+ mobilization in many types of cells. We examined the possible involvement of Edg‐3, one of the putative S1P receptors, in the phospholipase C (PLC)‐Ca2+ system. S1P increased the cytoplasmic free Ca2+ concentration without detectable inositol phosphate production in vector‐transfected CHO cells. In the Edg‐3‐transfected cells, however, the S1P‐induced Ca2+ response was clearly enhanced, which was associated with a significant production of inositol phosphate. These S1P‐induced responses in the Edg‐3‐transfected cells were inhibited by U73122, a potent PLC inhibitor. We conclude that Edg‐3 may be one of the S1P receptors participating in the activation of the PLC‐Ca2+ system.
A novel interaction mechanism accounting for different acylphosphatase effects on cardiac and fast twitch skeletal muscle sarcoplasmic reticulum calcium pumps
Authors: Nediani Chiara , Fiorillo Claudia , Rigacci Stefania , Magherini Francesca , Francalanci Michela , Liguri Gianfranco , Pacini Alessandra , Nassi Paolo
Published: July 2002

In cardiac and skeletal muscle Ca2+ translocation from cytoplasm into sarcoplasmic reticulum (SR) is accomplished by different Ca2+‐ATPases whose functioning involves the formation and decomposition of an acylphosphorylated phosphoenzyme intermediate (EP). In this study we found that acylphosphatase, an enzyme well represented in muscular tissues and which actively hydrolyzes EP, had different effects on heart (SERCA2a) and fast twitch skeletal muscle SR Ca2+‐ATPase (SERCA1). With physiological acylphosphatase concentrations SERCA2a exhibited a parallel increase in the rates of both ATP hydrolysis and Ca2+ transport; in contrast, SERCA1 appeared to be uncoupled since the stimulation of ATP hydrolysis matched an inhibition of Ca2+ pump. These different effects probably depend on phospholamban, which is associated with SERCA2a but not SERCA1. Consistent with this view, the present study suggests that acylphosphatase‐induced stimulation of SERCA2a, in addition to an enhanced EP hydrolysis, may be due to a displacement of phospholamban, thus to a removal of its inhibitory effect.
The DNA binding activity of Translin is mediated by a basic region in the ring‐shaped structure conserved in evolution
Authors: Aoki Katsunori , Suzuki Kenji , Ishida Reiko , Kasai Masataka
Published: July 2002

DNA binding proteins, for the most part, function as dimers or tetramers which recognize their target sequences. Here we show that Translin, a novel single‐stranded DNA end binding protein, forms a ring‐shaped structure conserved throughout evolution and that this structure is responsible for its DNA binding activity. Point mutations at Leu184 and Leu191 in the leucine zipper motif of human Translin resulted in loss of the multimeric structure and abrogation of DNA binding. Point mutations at R86, H88, H90 to T86, N88, N90 in one of the basic regions, however, completely inhibited the DNA binding activity without affecting the multimeric structure. These results support the view that the DNA binding domain of Translin is formed in the ring‐shaped structure in combination with its basic region (amino acids 86–97) polypeptides.
The localisation of 2‐carboxy‐d‐arabinitol 1‐phosphate and inhibition of Rubisco in leaves of Phaseolus vulgaris L
Authors: Parry Martin A.J , Andralojc P.J , Lowe Helen M , Keys Alfred J
Published: July 2002

A recent controversial report suggests that the nocturnal inhibitor of Rubisco, 2‐carboxy‐d‐arabinitol 1‐phosphate (CA1P), does not bind to Rubisco in vivo and therefore that CA1P has no physiological relevance to photosynthetic regulation. It is now proved that a direct rapid assay can be used to distinguish between Rubisco‐bound and free CA1P, as postulated in the controversial report. Application of this direct assay demonstrates that CA1P is bound to Rubisco in vivo in dark‐adapted leaves. Furthermore, CA1P is shown to be in the chloroplasts of mesophyll cells. Thus, CA1P does play a physiological role in the regulation of Rubisco.
Denatured states of human carbonic anhydrase II: an NMR study of hydrogen/deuterium exchange at tryptophan‐indole‐HN sites
Authors: Jonasson Per , Kjellsson Annika , Sethson Ingmar , Jonsson Bengt-Harald
Published: July 2002

Hydrogen/deuterium (H/D) exchange measurements in low and moderate concentrations of GuHCl were conducted on the side chain HN atoms of the seven tryptophans of pseudo wild‐type human carbonic anhydrase II. Tryptophans 5, 16 and 245, situated in or close to the N‐terminal domain were found to have little protection against exchange. The H/D exchange results for Trp‐123, Trp‐192 and Trp‐209 showed that a previously identified molten globule and the native state gave a similar protection against exchange. Global unfolding of the protein is necessary for the efficient exchange at Trp‐97, which is located in the central part of the β‐sheet.
Expression of plasminogen‐related gene B varies among normal tissues and increases in cancer tissues
Authors: Tateno Tadashi , Ichinose Akitada
Published: July 2002

We previously found that the promoter activity of plasminogen (PLG)‐related gene B (PRGB) was 5‐fold that of PLG. We have since examined the transcript levels of PRGB among various normal human tissues, and compared these findings with those of PLG. Reverse transcription‐PCR analysis revealed that the PRGB expression varied widely among different tissues, while PLG was expressed only in the liver and kidney. RNA samples obtained from cultured cell lines also demonstrated differing PRGB expression. Furthermore, increased PRGB expression was observed in several fresh samples of cancer tissue obtained from cancer patients when compared with surrounding normal tissues.
The chloroplast infA gene with a functional UUG initiation codon
Authors: Hirose Tetsuro , Ideue Takashi , Wakasugi Tatsuya , Sugiura Masahiro
Published: July 2002

All chloroplast genes reported so far possess ATG start codons and sometimes GTGs as an exception. Sequence alignments suggested that the chloroplast infA gene encoding initiation factor 1 in the green alga Chlorella vulgaris has TTG as a putative initiation codon. This gene was shown to be transcribed by RT‐PCR analysis. The infA mRNA was translated accurately from the UUG codon in a tobacco chloroplast in vitro translation system. Mutation of the UUG codon to AUG increased translation efficiency approximately 300‐fold. These results indicate that the UUG is functional for accurate translation initiation of Chlorella infA mRNA but it is an inefficient initiation codon.
Trans‐splicing of a voltage‐gated sodium channel is regulated by nerve growth factor
Authors: Akopian Armen N. , Okuse Kenji , Souslova Veronika , England Steven , Ogata Nobukino , Wood John N.
Published: July 2002

Mammalian sensory neurons express a voltage‐gated sodium channel named SNS. Here we report the identification of an SNS transcript (SNS‐A) that contains an exact repeat of exons 12, 13 and 14 encoding a partial repeat of domain II. Because the exons 12–14 are present in single copies in genomic DNA, the SNS‐A transcript must arise by trans‐splicing. Nerve growth factor, which regulates pain thresholds, and the functional expression of voltage‐gated sodium channels increases the levels of the SNS‐A transcript several‐fold both in vivo and in vitro as measured by RNase protection methods, as well as RT‐PCR. These data demonstrate a novel regulatory role for the nerve growth factor and are the first example of trans‐splicing in the vertebrate nervous system.
Increased oxidative stress in the RAW 264.7 macrophage cell line is partially mediated via the S‐nitrosothiol‐induced inhibition of glutathione reductase
Authors: Butzer Urwe , Weidenbach Hans , Gansauge Susanne , Gansauge Frank , Beger Hans G , Nussler Andreas K
Published: July 2002

We investigated whether endogenously or exogenously produced nitric oxide (NO) can inhibit cellular glutathione reductase (GR) via the formation of S‐nitrosothiols to decrease cellular glutathione (GSH) and increase oxidative stress in RAW 264.7 cells. The specificity of this inhibition was demonstrated by addition of a NO‐synthase inhibitor, and met‐ or oxyhemoglobin. Using isolated GR we found that only certain NO donors inhibit this enzyme via S‐nitrosothiol. Furthermore, we found that cellular GSH decrease is paralleled by an increase of superoxide anion production. Our results show that the GR enzyme is a potential target of S‐nitrosothiols to decrease cellular GSH levels and to induce oxidative stress in macrophages.
Lack of heat shock response triggers programmed cell death in a rat histiocytic cell line
Authors: Sreedhar A.S , Pardhasaradhi B.V.V , Begum Zareena , Khar Ashok , Srinivas Usha K
Published: July 2002

Stress response is a universal phenomenon. However, a rat histiocytic cell line, BC‐8, showed no heat shock response and failed to synthesize heat shock protein 70 (hsp70) upon heat shock at 42°C for 30 min. BC‐8 is a clone of AK‐5, a rat macrophage tumor line that is adapted to grow in culture and has the same chromosome number and tumorigenic potential as AK‐5. An increase in either the incubation temperature or time or both to BC‐8 cells leads to loss of cell viability. In addition, heat shock conditions activated apoptotic cell death in these cells as observed by cell fragmentation, formation of nuclear comets, apoptotic bodies, DNA fragmentation and activation of ICE‐like cysteine proteases. Results presented here demonstrate that BC‐8 cells cannot mount a typical heat shock response unlike all other eukaryotic cells and that in the absence of induction of hsps upon stress, these cells undergo apoptosis at 42°C.
A human homolog of Drosophila warts tumor suppressor, h‐warts, localized to mitotic apparatus and specifically phosphorylated during mitosis
Authors: Nishiyama Yasuyuki , Hirota Toru , Morisaki Tetsuro , Hara Toshihiro , Marumoto Tomotoshi , Iida Shin-ichi , Makino Keishi , Yamamoto Hideyuki , Hiraoka Takehisa , Kitamura Nobuo , Saya Hideyuki
Published: July 2002

We identified a human homolog of Drosophila warts tumor suppressor gene, termed h‐warts, which was mapped at chromosome 6q24‐25.1. The h‐warts protein has a serine/threonine kinase domain and is localized to centrosomes in interphase cells. However, it becomes localized to the mitotic apparatus, including spindle pole bodies, mitotic spindle, and midbody, in a highly dynamic manner during mitosis. Furthermore, h‐warts is specifically phosphorylated in cells at mitotic phase, most likely by Cdc2 kinase. These findings suggest that h‐warts functions as a component of the mitotic apparatus and is involved in proper progression of mitosis.
Smooth muscle α‐tropomyosin crosslinks to caldesmon, to actin and to myosin subfragment 1 on the muscle thin filament
Authors: Golitsina Nina L. , Lehrer Sherwin S.
Published: July 2002

To obtain proximity information between tropomyosin (Tm) and caldesmon (CaD) on the muscle thin filament, we cloned gizzard αTm and created two single Cys mutants S56C/C190S (56Tm) and D100C/C190S (100Tm). They were labeled with benzophenone maleimide (BPM) and UV‐irradiated on thin filaments. One chain of BPM‐56Tm and two chains of BPM‐100Tm crosslinked to CaD. Only BPM‐100Tm crosslinked to actin in the absence and presence of CaD and binding of low ratios of myosin subfragment 1 (S1) prevented the crosslinking. Tm‐S1 crosslinks were produced when actin·Tm was saturated with S1. Thus, CaD on the actin·Tm filament is located <10 Å away from Tm amino acids 56 and 100; in the closed state of the actin·Tm filament, Tm residue 100 is located close to the actin surface and is moved further away in the S1‐induced open state; in the open state, S1 binds close to Tm.
The teaching of evidence‐based reproductive health in developing countries
Authors: Olatunbosun O.A. , Edouard L.
Published: July 2002

Evidence‐based medicine is gaining increased importance in clinical practice. Unfortunately, prevailing circumstances have prevented this application of the best available evidence to clinical decision‐making for reproductive health in developing countries. Poor infrastructure, inadequate personnel, gross underfunding of services for health and education as well as a conservative education system are obstacles in effecting change. By examining these impediments, we suggest strategies for clinical education and research with the aim of improving reproductive health care in developing countries. Evidence‐based reproductive health will be promoted through access to appropriate information, interest from government and consumers, curriculum changes, application of research findings and finally, the judicious use of its principles in clinical practice.