bluuebunny/crossref_metadata_2025 huggingface dataset:
The Fidelity of Misinsertion and Mispair Extension Throughout DNA Synthesis Exhibited by Mutants of the Reverse Transcriptase of Human Immunodeficiency Virus Type 2 Resistant to Nucleoside Analogs
Authors: Taube Ran , Avidan Orna , Hizi Amnon
Published: June 2004

The AIDS‐causing retroviruses, human immunodeficiency virus types 1 and type 2 (HIV‐1 and HIV‐2, respectively) undergo extensive genetic variations, which effect their pathogenesis and resistance to drug therapy. It was postulated that this genetic hypervariability results from high rates of viral replication in conjugation with a relatively low fidelity of DNA synthesis [typical to the reverse transcriptases (RT) of these retroviruses]. As part of studying structure/function relationship in HIV RT, mutational analyses were conducted to identify amino acid residues which are involved in affecting the fidelity of DNA synthesis. The formation of 3′‐mispaired DNA due to nucleotide misinsertions, and the subsequent elongation of this mismatched DNA were shown to be major determinants in affecting those substitutions during DNA synthesis (exhibited in vitro by HIV RT). It was interesting to find a correlation between sensitivity to nucleoside analogs (due to the ability to incorporate or reject an incoming analog) and the fidelity of DNA synthesis (which depends on the capacity to incorporate and extend a wrong nucleotide). Such a connection has already been found for several drug‐resistant mutants of HIV‐1 RT, with an increased fidelity of DNA synthesis relative to the wild‐type RT.

In the present study we have examined the fidelity of DNA synthesis using the same parameters of misinsertion and mispair extension for five novel drug‐resistant mutants of HIV‐2 RT; i.e. the single mutants [Val74]RT, [Gly89]RT and [Tyr215]RT and the double mutants [Val74, Tyr215]RT and [Gly89, Tyr215]RT. This comparative study suggests that unlike the Val74 mutant of HIV‐1 RT, which was shown earlier to display a substantially enhanced fidelity, the comparable mutant of HIV‐2 RT has fidelity similar to that of the wild‐type RT. Depending on the assay employed and the DNA sequences extended, most other mutants of HIV‐2 RT display moderate effects on the enzyme, leading to mild increases in fidelity of DNA synthesis. This implies a more complex and less distinctive correlation between drug‐resistance, misinsertion and mispair extension in HIV‐2 RT in contrast to HIV‐1 RT, providing evidence for potential biochemical differences between these two related RT.
The Histone H1 Genes of the Dipteran Insect, Chironomus thummi, Fall under Two Divergent Classes and Encode Proteins with Distinct Intranuclear Distribution and Potentially Different Functions
Authors: Trieschmann Lothar , Schulze Ekkehard , Schulze Bettina , Grossbach Ulrich
Published: June 2004

Four histone H1 genes of the midge, Chironomus thummi piger, and three H1 genes of the subspecies C. thummi thummi have been cloned and assigned to the four different H1 proteins from C. thummi larvae. Together with an earlier cloned H1 gene from C. thummi thummi [Hankeln, T. & Schmidt, E. R. (1991) Chromosoma 101, 25–31], these genes probably constitute the complete complement of H1 genes in both subspecies. They were found to fall under two classes that differ remarkably in their gene copy numbers, genomic organization, structure of flanking sequences, codon usage, and expression during embryonic development, and that encode H1 proteins of divergent structure. Histone H1 I‐1 contains an inserted sequence, KAPKAPKAPKSPKAE in C. thummi piger, and KAPKAPKSPKAE in C. thummi thummi, that is lacking in the other H1 variants, H1 II‐1, H1 II‐2, and H1 III‐1. In the immediate neighbourhood to the inserted sequence, a substitution in the H1 I‐1 protein sequence dramatically enhances the potential to form a reversed turn. In early development, H1 I‐1 is expressed at a higher rate than the other H1 genes. The transcripts have a size of about 1 kb; in addition, the H1 I‐1 gene exhibited two minor transcripts of about 2.5 and > 3 kb size in middle blastoderm that are possibly polyadenylated. Together with our earlier finding that histone H1 I‐1 is found in a limited number of polytene chromosome bands whereas the other H1 histones are uniformly distributed in chromatin, these results intimate functional differences between the two classes of H1 genes and their products.
Gene Analysis of an NADP‐Linked Isocitrate Dehydrogenase Localized in Peroxisomes of the n‐Alkane‐Assimilating Yeast Candida tropicalis
Authors: Kawachi Hiroyuki , Shimizu Kaori , Atomi Haruyuki , Sanuki Shin‐ichi , Ueda Mitsuyoshi , Tanaka Atsuo
Published: June 2004

In n‐alkane‐utilizing yeast, Candida tropicalis, two NADP‐linked isocitrate dehydrogenase (NADP‐IDH) isozymes are present, one in mitochondria (Mt‐NADP‐IDH) and the other in peroxisomes (Ps‐NADP‐IDH). Here we report the isolation, sequencing, and expression of the gene encoding Ps‐NADP‐IDH (CtIDP2), distinct from the Mt‐NADP‐IDH gene (CtIDP1). Based on the N‐terminal amino acid sequence of purified Ps‐NADP–IDH, a cDNA fragment specific for Ps‐NADP–IDH was obtained by the 5′‐RACE method. Using this fragment as a probe, the genomic CtIDP2 gene was isolated. Nucleotide sequence analysis of CtIDP2 disclosed that the region encoding CtIdp2p had a length of 1233 bp, corresponding to 411 amino acid residues. The deduced N‐terminal amino acid sequence matched the results obtained from the purified protein. When this CtIDP2 was expressed in Saccharomyces cerevisiae using the C. tropicalis isocitrate lyase gene promoter (UPR‐ICL), high intracellular NADP–IDH activity was observed. Comparison of amino acid sequences and phylogenetic tree analysis with NADP–IDH enzymes from all reported eukaryotic sources revealed that mammalian mitochondrial NADP–IDHs formed a cluster, as did plant NADP–IDHs. CtIdp2p and other yeast NADP–IDHs were not included in these clusters and seemed to diverge at an early stage from all other enzymes of higher eukaryotes. Ps‐NADP–IDH had no typical C‐terminal peroxisomal targeting signal and no processing was demonstrated at the N‐terminus. However, we could find a region near the N‐terminus of the protein with high similarity to both the putative N‐terminal peroxisomal targeting signal sequence of Fox3p of S. cerevisiae and an internal region of Pox4p of C. tropicalis. The results of northern blot analysis indicated that the biosynthesis of CtIdp2p was induced in a medium containing alkanes as a carbon source, where profuse proliferation of peroxisomes is observed.
Expression, Reconstitution and Characterization of Prolixin‐S as a Vasodilator
Authors: Yuda Masao , Higuchi Kuniko , Sun Jianxin , Kureishi Yasuko , Ito Masaaki , Chinzei Yasuo
Published: June 2004

Prolixin‐S, an anticoagulant from the salivary gland of the blood‐sucking insect Rhodnius prolixus is also one of the members of salivary gland hemoproteins. We produced recombinant protein using a baculovirus‐insect cell expression system, reconstituted the hemoprotein and made some characterization of it as a nitric oxide carrier. The reconstituted protein exhibited the absorption spectrum of a high‐spin ferric hemoprotein with a Soret absorption peak at 400 nm. By binding nitric oxide (NO‐prolixin‐S), the Soret band shifted from 400 nm to 420 nm and two sharp bands (Q bands, at 535 nm and 565 nm) also appeared in the visible region. In a bioassay with aortic smooth muscle, NO‐prolixin‐S showed strong relaxation activity in a dose‐dependent manner, which demonstrated that prolixin‐S really acts as an NO carrier. A Soret absorption change also indicated that nitric oxide was gradually released under these conditions (pH 7.4 and 37°C). However, at low temperature (20°C) and/or low pH (pH 6), which mimic those in the insect's salivary glands, the releasing became very slow. These different NO‐binding properties would enable prolixin‐S to reserve nitric oxide in the salivary glands and release it in the host's blood vessels.
The 11β‐Hydroxysteroid Dehydrogenase System, A Determinant of Glucocorticoid and Mineralocorticoid Action
Authors: Maser Edmund , Oppermann Udo C. T.
Published: June 2004

Carbonyl reduction is a significant step in the biotransformation leading to the elimination, of endogenous and exogenous aldehydes, ketones and quinones. This reaction is mediated by members of the aldoketo reductase and short‐chain dehydrogenase/reductase (SDR) superfamilies. The essential role of these enzymes in protecting organisms from damage by the accumulation of toxic carbonyl compounds is generally accepted, although their physiological roles are not always clear. Recently, the SDR enzyme 11β‐hydroxysteroid dehydrogenase‐1 has been identified to perform an important role in the detoxification of non‐steroidal carbonyl compounds, in addition to metabolising its physiological glucocorticoid substrates. This review summarises the current knowledge of type‐1 11β‐hydroxysteroid dehydrogenase and discusses possible substrate/inhibitor interactions. They might impair either the physiological function of glucocorticoids or the detoxification of non‐steroid carbonyl compounds.
Different Consequences of Incorporating Chloroplast Ribosomal Proteins L12 and S18 into the Bacterial Ribosomes of Escherichia Coli
Authors: Weglöhner Wolfgang , Jünemann Ralf , Von Knoblauch Klaus , Subramanian Alap R.
Published: June 2004

We have incorporated chloroplast ribosomal proteins (R‐proteins) L12 and S18 into Escherichia coli ribosomes and examined the hybrid ribosomes for their ability to form polysomes in vivo and perform poly(U)‐dependent poly(Phe) synthesis in vitro. The rye chloroplast S18 used for the experiment is a highly divergent protein (170 amino acid residues; E. coli S18, 74 residues), containing a repeating, chloroplast‐specific, heptapeptide motif, and has amino acid sequence identity of only 35% to E. coli S18. When expressed in E. coli, chloroplast S18 was assembled in E. coli ribosomes. The latter formed polysomes in vivo at about the same rate as the host ribosomes, indicating that the replacement of E. coli S18 with its chloroplast homologue has only a minor, if any, effect on function. The L12 protein is much more conserved in sequence and chain length, and is known to have a very important function. The Arabidopsis chloroplast L12 used in the experiment was incorporated into E. coli 50S subunits that associated with the 308 subunits to form ribosomes, but the latter were unable to form polysomes. This result indicates functional inactivation of E. coli ribosomes by a chloroplast R‐protein. To further confirm this result, we overproduced chloroplast L12 through the use of a secretion vector and purified the protein to homogeneity. Chloroplast L12 could be efficiently incorporated in vitro into L7/12‐lacking E. coli ribosomes, but the hybrid ribosomes were totally inactive in poly(U)‐dependent poly(Phe) synthesis. Computer modeling of the spatial structure of all known chloroplast L12 proteins (using E. coli L12 coordinates) indicated a ‘chloroplast loop’ present only in chloroplast L12. The presence of this loop might have a role in the observed inactivation. Taken together with previously reported results (summarized in this paper), it would appear that the features of chloroplast R‐proteins concerned with specific functions are more divergent than their assembly properties.

We have previously described methods suitable for overproduction and purification of chloroplast R‐proteins that are encoded in organellar DNA (≈20), but that gave poor yield for those encoded in the nuclear DNA (≈45). Here we describe a method that overcomes this problem and allows the purification of nucleus‐encoded chloroplast R‐proteins in milligram quantities.
Cloning and Characterization of the Arginine‐Specific Carbamoyl‐Phosphate Synthetase from Bacillus stearothermophilus
Authors: Yang Hsiuchin , Park Seung‐Moon , Nolan William G. , Lu Chung‐Dar , Abdelal Ahmed T.
Published: June 2004

Bacillus stearothermophilus contains two carbamoyl‐phosphate synthetases (CPS), one specific for pyrimidine biosynthesis and the other for arginine biosynthesis. The pyrimidine‐specific CPS is repressed by exogenous pyrimidines, and its activity is inhibited by UMP and activated by 5‐phospho‐a‐D‐ribosyl diphosphate. The arginine‐specific CPS is similarly repressed by exogenous arginine but its activity is not sensitive to these or other potential effectors. Each of the two enzymes consist of two unequal subunits, as is the case for other microbial CPS; however, the large subunit for the arginine‐specific CPS is smaller than that for the pyrimidine‐specific enzyme. Comparison of the derived amino acid sequence for the cloned large subunit of the arginine‐specific CPS with those for subunits from pyrimidine‐sensitive CPS showed significant similarity throughout the polypeptides except at the carboxy terminus, which was identified by other laboratories to contain the binding site for the pyrimidine effector. Unlike the results previously reported for CPS from an enteric mesophile, the kinetic properties of the arginine‐specific CPS were not affected by growth of B. stearothermophilus at temperatures near the minimal growth temperature. Furthermore, calorimetric studies showed that the thermal stability of cloned CPS was identical regardless of the growth temperature of B. stearothermophilus between 42°C and 63°C. The thermal stability of cloned CPS was not affected by expression at 37°C in Bacillus subtilis or Escherichia coli. In contrast, the thermal stabilities for CPS and other proteins were higher in extracts of cells grown at higher temperatures. These results indicate that cellular factors, probably chaperonins, are necessary for thermal stability of proteins at and below the optimal temperature for this thermophile.
Topological Photoaffinity Labeling of the Rabbit Ileal Na+/Bile‐Salt‐Cotransport System
Authors: Kramer Werner , Wess Günther , Bewersdorf Ulrike , Corsiero Daniel , Girbig Frank , Weyland Claudia , Stengelin Siegfried , Enhsen Alfons , Bock Klaus , Kleine Horst , Le Dreau Marie‐Anne , Schäfer Hans‐Ludwig
Published: June 2004

For the investigation of the topology of the rabbit ileal Na+/bile‐salt‐cotransport system, composed of a 93‐kDa integral membrane protein and a peripheral 14‐kDa bile‐acid‐binding protein (ILBP), we have synthesized photolabile dimeric bile‐salt‐transport inhibitors (photoblockers), G1‐X‐G2, where two bile acid moieties (G1 and G2) are tethered together via a spacer, X. and where one of the two bile acid moieties carries a photoactivatable group. These photoblockers specifically interact with the ileal Na+/bile‐saltcotransport system as demonstrated by a concentration‐dependent inhibition of [3H]cholyltaurine uptake by rabbit ileal brush‐border membrane vesicles and by inhibition of photolabeling of the 93‐kDa and 14‐kDa bile‐salt‐binding proteins by 7,7‐azo and 3,3‐azo derivatives of cholyltaurine. Ileal bile‐salt uptake was specifically inhibited by the photoblockers, which were not taken up themselves by the small intestine as demonstrated by in vivo ileal perfusion.

Dependent on the photoblocker used several polypeptides in the molecular‐mass range of 14–130 kDa were labeled. The cytoplasmically attached 14‐kDa ILBP was significantly labeled only by inhibitors that are photoactivatable in bile acid moiety G1, suggesting that during binding and translocation of a bile‐salt molecule by the ileal bile‐salt‐transport system the steroid nucleus gets access to the cytoplasmic site of the ileal brush‐border membrane first. Photoaffinity labeling in the frozen state with the transportable 3,3‐azo and 7,7‐azo derivatives of cholyltaurine revealed a time‐dependent increase in the extent of labeling of the 14‐kDa and 93‐kDa proteins, suggesting a labeling of these proteins from the cytoplasmic site of the ileal brush‐border membrane. By photoaffinity labeling in the frozen state with the various photoblockers time‐dependent changes in the extent of photoaffinity labeling of bile‐salt‐binding proteins were observed, demonstrating the possibility of topological analysis of the rabbit ileal Na+/bile‐saltcotransport system.
DNA Methylation Accounts for the Inhibition of Collagen VI Expression in Transformed Fibroblasts
Authors: Kopp Martin U. , Winterhalter Kaspar H. , Trueb Beat
Published: June 2004

The expression of collagen VI, an adhesive glycoprotein of the extracellular matrix, is completely inhibited in virally transformed fibroblasts and in many cell lines derived from spontaneous mesenchymal tumors. Here we present evidence that DNA methylation plays an important role in this inhibition: (a) The mRNA level for DNA methyltransferase is highly increased in simian virus 40 (SV40)–transformed fibroblasts compared with normal cells and this increase correlates with the decrease of the mRNA level for collagen VI. (b) Methylation of the α2(VI) collagen promoter in vitro abolishes promoter activity in a transient transfection assay, (c) Genomic sequencing reveals extensive methylation of the promoter region in SV40‐transformed cells, but virtually no methylation of the corresponding region in normal cells. Increased methylation is also observed in a rhabdomyosarcoma cell line, (d) Two of the cis‐acting elements of the α2(VI) collagen promoter lose their affinity for transcription factor AP2 when methylated in vitro as demonstrated by gel retardation experiments. DNA methylation is therefore involved in the silencing of the α2(VI) collagen gene. It seems likely that the same mechanism is also responsible for the repression of other transformation‐sensitive proteins.
Dimerization of the Synaptic Vesicle Protein Synaptobrevin (Vesicle‐Associated Membrane Protein) II Depends on Specific Residues within the Transmembrane Segment
Authors: Laage Rico , Langosch Dieter
Published: June 2004

Synaptobrevin is an integral membrane protein of presynaptic vesicles and is essential for neurotransmitter release. Previously, a dimeric quaternary structure has been proposed by cross‐linking experiments performed on brain fractions. Here, we demonstrate that heterologously expressed and solubilized synaptobrevin II forms a homodimer. The dimers were detected upon cross‐linking with a homobifunctional lysine‐reactive reagent or by oxidation of the single cysteine residue located within the transmembrane segment. Dimerization was also observed without prior cross‐linking upon SDS/PAGE under mild conditions. Interestingly, dimerization required the presence of the transmembrane segment which therefore is inferred to be the principal site of subunit‐subunit interaction. The residues comprizing this segment were individually mutated. Dimerization of some point mutants was significantly impaired, which proved the sequence specificity of interaction and identified residues contributing to the subunit‐subunit interface. The distribution of these residues (Leu99, IIe102, Cys103, Leu107, IIe110, and IIe111) suggests that the transmembrane segment has an α‐helical structure and that the helices pair in a right‐handed fashion. The importance of the transmembrane segment for subunit‐subunit interaction relates synaptobrevin to fusogenic membrane proteins of enveloped viruses where transmembrane segments have been implicated in both oligomerization and membrane fusion.
cDNA Cloning of a Trichoderma reesei Cellulase and Demonstration of Endoglucanase Activity by Expression in Yeast
Authors: Saloheimo Markku , Nakari‐SetäLä Tiina , Tenkanen Maija , Penttilä Merja
Published: June 2004

A Trichoderma reesei cDNA encoding a previously unknown protein with a C‐terminal cellulosebinding domain was obtained by complementation screening of a T. reesei cDNA library in a sec1 yeast mutant impaired in protein secretion. The T. reesei protein shows amino acid similarity over its entire length to the Agaricus bisporusi cellulose‐induced protein CEL1 whose function is not known. These two proteins form a new glycosyl hydrolase family, number 61. Expression of the T. reesei cDNA in yeast showed that it encoded a protein with endoglucanase activity and thus the protein was named EGIV and the corresponding gene eg14. Polyclonal antibodies were prepared against EGIV produced in Escherichia coli and detected a 56‐kDa protein in the T. reesei culture supernatant. Northern hybridisation revealed that T. reesei egl4 is regulated in the same manner as other cellulase genes of this fungus.
Some Details of the Reaction Mechanism of Glucoamylase from Aspergillus Niger— Kinetic and Structural Studies on Trp52→Phe and Trp317→Phe Mutants
Authors: Christensen Trine , Stopper Bjarne B. , Svensson Birte , Christensen Ulla
Published: June 2004

Presteady and steady‐state kinetic results on the interactions of a wild‐type, and the mutant glucoamylases Trp52→Phe and Trp317→Phe, from Aspergillus niger with maltose, maltotriose and maltotetraose have been obtained and analyzed. The results are compared with previous ones on the mutants, Trpl20→Phe and Glu180→Gln, and with results obtained from structure energy minimization calculations based on known three‐dimensional structural data. All results are in accordance with a three‐step reaction model involving two steps in the substrate binding and a rate‐determining catalytic step. Trp317 and Glu180 belong to different subsites, but are placed on the same flank of the active site (β‐flank). The Trp317→Phe and the Glu180→Gln mutants show almost identical kinetic results: weakening of the substrate binding, mainly caused by changes in the second reaction step, and practically no change of the catalytic rate. Structure energy minimization calculations show that the same loss of Arg305 and Glu180 hydrogen bonds to the substrate occurs in the Michaelis complexes of each of these mutants. These results indicate that important interactions of the active site may be better understood from a consideration of its flanks rather than of its subsites. The results further indicate differences in the substrate binding mode of maltose and of longer substrates. Trp52 and Trp120 each interact with the catalytic acid, Glu179, and are placed on the flank (α‐flank) of the active site opposite to Trp317, Arg305 and Glu180. Also the Trp52→Phe and Trp120→Phe mutants show kinetic results similar to each other. The catalytic rates are strongly reduced and the substrates are bound more strongly, mainly as a result of the formation of a more stable complex in the second reaction step. All together, the substrate binding mechanism seems to involve an initial enzyme‐substrate complex, in which the β‐flank plays a minor role, except for maltose binding; this is followed by a conformational change, in which hydrogen bonds to Arg305 and Glu180 of the β‐flank are established and the correct alignment on the α‐flank of Glu179, the general acid catalyst, governed by its flexible interactions with Trp52 and Trp120, occurs.
Membrane‐Type Matrix Metalloproteinases 1 and 2 Exhibit Broad‐Spectrum Proteolytic Capacities Comparable to Many Matrix Metalloproteinases
Authors: D'ortho Marie‐Pia , Will Horst , Atkinson Susan , Butler Georgina , Messent Anthea , Gavrilovic Jelena , Smith Bryan , Timpl Rupert , Zardi Luciano , Murphy Gillian
Published: June 2004

Soluble proenzyme forms of the catalytic domains of membrane‐type matrix metalloproteinases 1 and 2 (MT1‐MMP and MT2‐MMP) and a form of MTI‐MMP containing the catalytic and hemopexin domains were expressed as soluble recombinant proteins. Purified, activated forms of the MT‐MMP were shown to degrade fibronectin, tenascin, nidogen, aggrecan and perlecan. Only MT2‐MMP showed activity against laminin. MT1‐MMP retaining the hemopexin domain was able to specifically cleave native type‐I and type‐III collagens into the 3/4‐1/4 fragments typical of the specific collagenases. The catalytic domain alone did not retain this activity. The MT‐MMP did not degrade interleukin‐1β, but, similarly to many other MMP, could process a pro [tumor necrosis factor (TNF) α] fusion protein to release mature TNF. However, the latter was subsequently degraded into smaller fragments. These results demonstrate that, in addition to their ability to activate other MMP, such as progelatinase A/proMMP2 and procollagenase‐3/proMMP13, MT‐MMP degrade a number of extracellular matrix macromolecules. Their location at the surface of cells implies that they could play a significant role in the modulation of cell‐matrix interactions.
Identification of the Active Site Histidine in the Corrinoid Protein MtrA of the Energy‐Conserving Methyltransferase Complex From Methanobacterium Thermoautotrophicum
Authors: Harms Ulrike , Thauer Rudolf K.
Published: June 2004

The energy‐conserving corrinoid‐containing MtrA‐H complex from Methanobacterium thermoautotrophicum is composed of eight different subunits of which MtrA harbors the corrinoid prosthetic group. EPR spectroscopic evidence has recently been presented for a histidine residue as a cobalt ligand of the cobamide [Harms, U. & Thauer, R. K. (1996a) Eur J. Biochem. 241, 149–154]. This active site histidine was now identified by site‐directed mutagenesis to be His84 in the MtrA sequence that contains three histidines. This result was substantiated by sequence comparison of MtrA froin M. thermoautotrophicum, Methanococcus jannaschii, and Methanopyrus kandleri and of MtxA from Methanosarcina barkeri showing that only His84 is conserved. For comparison, the DNA sequences of the mtrEDCBAGH operon in M. kundleri and of the mtxXAH operon in M. barkeri were determined.
A DNA‐Topoisomerase‐11–Binding Protein with Eight Repeating Regions Similar to DNA‐repair Enzymes and to a Cell‐Cycle Regulator
Authors: Yamane Kazuhiko , Kawabata Masahiro , Tsuruo Takashi
Published: June 2004

A two‐hybrid system was used to isolate factors that interact with the C‐terminal region of DNA topoisomerase IIβ. A positive clone isolated from a HeLa cDNA library encoded 1.522 amino acid residues (molecular mass 170670). The protein, designated topoisomerase‐IIβ‐binding protein 1 (TopBP1), interacted with the C‐terminal region of topoisomerase IIβ synthesized in vitro. A database search indicated that TopBP1 possessed eight regions similar to regions of Rad4, Cut.5, Ect2, Revl and X‐ray repair cross‐complementing 1 (XRCC1) proteins and a region similar to auto‐modification sites of poly(ADP ribose) polymerase, suggesting that TopBP1 supported catalytic reactions of opoisomerase II through transient breakages of DNA strands.
Intracellular Distribution of an Integral Nuclear Pore Membrane Protein Fused to Green Fluorescent Protein — Localization of a Targeting Domain
Authors: Soderqvist Henrik , Imreh Gabriela , Kihlmark Madeleine , Linnman Carolina , Ringertz Nils , Hallberg Einar
Published: June 2004

The 121–kDa pore membrane protein (POM121) is a bitopic integral membrane protein specifically located in the pore membrane domain of the nuclear envelope with its short N‐terminal tail exposed on the luminal side and its major C‐terminal portion adjoining the nuclear pore complex. In order to locate a signal for targeting of POM121 to the nuclear pores, we over expressed selected regions of POM121 alone or fused to the green fluorescent protein (GFP) in transiently transfected COS‐1 cells or in a stably transfected neuroblastoma cell line. Microscopic analysis of the GFP fluorescence or immunostaining was used to determine the intracellular distribution of the over expressed proteins. The endofluorescent GFP tag had no effect on the distribution of POM121, since the chimerical POM121–GFP fusion protein was correctly targeted to the nuclear pores of both COS‐1 cells and neuroblastoma cells. Based on the differentiated intracellular sorting of the POM121 variants, we conclude that the first 128 amino acids of POM121 contains signals for targeting to the continuous endoplasmic reticuludnuclear envelope membrane system but not specifically to the nuclear pores and that a specific nuclear pore targeting signal is located between amino acids 129 and 618 in the endoplasmically exposed portion of POM121.
Effects of Brefeldin A on Phosphatidylcholine Phospholipase D and Inositolphospholipid Metabolism in HL‐60 Cells
Authors: Guillemain Isabella , Exton John H.
Published: June 2004

The involvement of the small GTP‐binding protein ADP‐ribosylation factor (ARF) in guanosine 5′‐[γ‐thio]triphosphate (GTP[S])‐dependent activation of phospholipase D (PLD) in HL‐60 cells has been well established in vitro. In this study, we tested the effect of brefeldin A, which prevents ARF activation by inhibiting guanine‐nucleotide‐exchange activity, on PLD stimulation by receptor agonists (formyl‐Met‐Leu‐Phe and ATP) and by the phorbol ester phorbol 12‐myristate 13‐acetate (PMA) in differentiated HL‐60 cells. However, brefeldin A did not affect the activation of PLD at a time (1 h) when it eliminated the activity of the trans‐Golgi enzyme galactosyltransferase. It also did not inhibit PLD activity in Golgienriched membranes treated with GTP[S] with or without ARF in vitro. However, longer times of brefeldin A treatment (> 6 h), progressively and completely inhibited the activation of PLD by formyl‐Met‐Leu‐Phe and partly inhibited (∼50%) the activation by PMA. In contrast, long‐term brefeldin A treatment did not inhibit the effect of GTP[S] on PLD in permeabilized HL‐60 cells. Long‐term brefeldin A treatment completely inhibited inositol phosphate production in response to formyl‐Met‐Leu‐Phe and ATP, indicating that it affected inositolphospholipid‐specific phospholipase C activity. These data indicate that the rapid inhibitory effect of brefeldin A on Golgi function is not associated with inhibition of receptor‐mediated or PMA‐mediated PLD activation in HL‐60 cells. However, longer‐term effects, presumably arising from the disruption of the Golgi, lead to a total inhibition of agonist activation of PLD and inositolphospholipid‐specific phospholipase C. In summary, these results do not support a role for brefeldin‐A‐sensitive ARF in agonist regulation of PLD in HL‐60 cells.
Structural Properties of Chimeric Peptides Containing a T‐cell Epitope Linked to a Fusion Peptide and their Importance for in Vivo Induction of Cytotoxic T‐cell Responses
Authors: Lelièvre Dominique , Hsu Shiou‐Chih , Daubos Philippe , Favard Cyril , Vigny Paul , Trudelle Yves , Steward Michael W. , Delmas Agnès
Published: June 2004

We have previously shown that when administered to mice without adjuvant, a chimeric peptide consisting of the fusion peptide F from measles virus protein linked at the C‐terminus of a cytotoxic T‐cell epitope from the M2 protein of respiratory syncytial virus efficiently primes for an major histocompatibility complex (MHC) class‐I restricted cytotoxic T lymphocyte (CTL) response. In this report, we demonstrated by microspectrofluorometry that the fusion‐peptide moiety bound to the plasma membrane of living cells. When the fusion peptide was linked to the C‐terminus of the CTL epitope, the chimeric peptide (M2–F) adopted a marked β‐sheet conformation. In contrast, when the fusion peptide was linked to the N‐terminus of the T‐cell epitope (F–M2), the chimeric peptide adopted an a‐helical conformation in the presence of trifluoroethanol. The immunogenicity of the two chimeric peptides for class‐I restricted CTL was also significantly different, the one adopting the a‐helical conformation being more immunogenic. Probably due to its obvious conversion to an α‐helical conformation, the F–M2 peptide could have a higher propensity to insert into membranes, as shown by microspectrofluorometry, with a resultant better immunogenicity than the M2–F peptide.
The BH3 Domain of Bax is Sufficient for Interaction of Bax with itself and with other Family Members and it is Required for Induction of Apoptosis
Authors: Simonen Marjo , Keller Hansjörg , Heim Jutta
Published: June 2004

bax is an apoptosis‐inducing member of the bcl‐2 multigene family. We have studied interactions of human Bax with itself, and with the apoptosis‐preventing members Bcl‐2 and Bcl‐xL using a yeast twohybrid system. Exhaustive Bax truncations were constructed and their interactions with full‐length family members studied. Bax interacted similarly with itself as with the apoptosis‐suppressing family members Bcl‐2 and Bcl‐xL in quantitative two‐hybrid studies. A region of 41 amino acids covering the recently discovered BH3 domain of Bax was found to be necessary and sufficient for all interactions of Bax. Bax truncations containing BH3, but lacking BH1 and BH2 homology domains, interacted with the other family members markedly more strongly than full‐length Bax, which may reflect conformational changes required for the interactions of full‐length Bax. The minimum requirements for Bax homodimerization were found to be the BH3 domain from one Bax molecule and a region covering BH3 plus BH1 from another. We also studied the apoptosis‐inducing activity of the Bax truncations upon microinjection of expression plasmids into rat fibroblasts. The BH3 region was required for the apoptosis‐inducing activity of Bax, whereas BH1, BH2 and the N‐terminus of Bax were dispensable.
Substrate Specificity of Cathepsins D and E Determined by N‐Terminal and C‐terminal Sequencing of Peptide Pools
Authors: Arnold Danièle , Keilholz Wieland , Schild Hansjörg , Dumrese Tilman , Stevanović Stefan , Rammensee Hans‐Georg
Published: June 2004

Degradation of protein antigens by cellular proteases is a crucial step in the initiation of a T‐cell‐mediated immune response. But still little is known about the enzymes responsible for the processing of antigens, including their specificity. In this paper, we show that the combination of automated N‐terminal sequencing with a newly developed method for C‐terminal sequencing of peptide pools generated by the aspartic proteases cathepsins D and E is a fast and easy method to obtain detailed information of the substrate specificity of these endopeptidases. Using a 15‐residue synthetic peptide library and a native protein as substrates, we confirm and extend the knowledge about the cleavage motif of cathepsin E where positions P1 and P1’ of the substrate must be occupied exclusively by hydrophobic amino acids with aromatic or aliphatic side chains. However, Val and Ile residues are not allowed at position P1. Position P2’ accepts a broad range of amino acids, including charged and polar ones. Additional requirements concerning the substrate positions P3’ and P4’ were also defined by pool sequencing. Furthermore, pool sequencing analysis of melittin digests with the aspartic proteases cathepsin D and E provided evidence that both enzymes share the same cleavage motif, identical to the one derived from the peptide library and the native protein. Therefore, pool sequencing analysis is a valuable and fast tool to determine the substrate specificity of any endopeptidase.
A Monoclonal Antibody Induces Opening of a Coiled Coil
Authors: Dürr Eberhard , Bosshard Hans R.
Published: June 2004

Antibody specificity is limited, and different antigens may cross‐react to varying degrees with the same antibody. An example is monoclonal antibody 42PF elicited against a 29‐residue random‐coil pep‐tide. 42PF cross‐reacts with a triple‐stranded coiled coil of related amino acid sequence, and there was circumstantial evidence for an antibody‐induced opening of the coiled coil [Leder, L., Berger, C., Bornhauser, S., Wendt, H., Ackermann, F., Jelesarov, I. & Bosshard, H. R. (1995) Biochemistry 34, 16509–16518]. To reveal in a direct way that antibody 42PF induces the opening of the coiled coil, we have compared the rates of H/D exchange of amide protons in the free and in the antibody‐bound coiled‐coil peptide using electrospray‐ionization mass spectrometry for the analysis of deuterium incorporation. Complete H/D exchange lasted several days in the tightly folded coiled‐coil conformation while in the presence of antibody 42PF the overall exchange took only minutes, corresponding to a thousand‐fold decrease of protection of the most slowly exchanging amide hydrogens of the folded coiled coil. This demonstrates that the antibody induced a considerable opening of the coiled coil.
Cell Respiration is Controlled by ATP, an Allosteric Inhibitor of Cytochrome‐c Oxidase
Authors: Arnold Susanne , Kadenbach Bernhard
Published: June 2004

The activity of cytochrome‐c oxidase, the terminal enzyme of the mitochondria) respiratory chain, is known to be regulated by the substrate pressure, i.e. the ferro‐/ferricytochrome c ratio, by the oxygen concentration, and by the electrochemical proton gradient ΔμH+ across the inner mitochondrial membrane. Here we describe a further mechanism of ‘respiratory control’ via allosteric inhibition of cytochrome‐c oxidase by ATP, which binds to the matrix domain, of subunit IV. The cooperativity between cytochrome‐c‐binding sites in the dimeric enzyme complex is mediated by cardiolipin, which is essential for cooperativity of the enzyme within the lipid membrane.
The 11β‐Hydroxysteroid Dehydrogenase System, A Determinant of Glucocorticoid and Mineralocorticoid Action
Authors: Seckl Jonathan R. , Chapman Karen E.
Published: June 2004

11β‐Hydroxysteroid dehydrogenases (11β‐HSD) catalyse the interconversion of active glucocorticoids (cortisol, corticosterone) and their inert 11‐keto derivatives (cortisone, 11‐dehydrocorticosterone). The type‐2 isozyme (11β‐HSD‐2) is a high‐affinity dehydrogenase that catalyses the rapid inactivation of glucocorticoids, thus ensuring selective access of aldosterone to otherwise non‐selective mineralocorticoid receptors in the distal nephron. Mutations of the gene encoding 11β‐HSD‐2 are responsible for the syndrome of apparent mineralocorticoid excess, in which cortisol illicitly occupies mineralocorticoid receptors, causing hypertension and hypokalaemia. 11β‐HSD‐2 is also highly expressed in the placenta and mid‐gestation fetus, where it may protect developing tissues from the often deleterious actions of glucocorticoids upon fetal growth and organ maturation. β‐HSD‐1 is probably an 11β‐reductase in vivo. Its function is obscure, but may amplify glucocorticoid action during the diurnal nadir, drawing upon the substantial circulating levels of 11‐keto steroids. Both isozymes are regulated during ontogeny and by a series of hormonal and other factors. 11β‐HSD provide an important control of glucocorticoid action at a cellular level, and may represent new targets for therapeutic intervention.
Cloning of a Putative G‐Protein‐Coupled Receptor from Arabidopsis thaliana
Authors: Josefsson Lars‐Göran , Rask Lars
Published: June 2004

We have cloned and characterized a cDNA from Arabidopsis thaliana that most likely encodes a novel member of the vast superfamily of G‐protein‐coupled receptor proteins (GPCRs). By taking advantage of amino acid sequence similarities between plant expressed sequence tags (ESTs) and established G‐proteincoupled receptor sequences, a probe was obtained which was used for the screening of an Arabidopsis cDNA library. The cDNA which was found is very infrequently represented in the cDNA library, suggesting a low and/or spatially restricted expression. A region of the translated sequence of the cDNA shows the highest similarity to CAMP receptors from the slime mold Dictyostelium discoideum. The same region is also similar to that in members of the animal calcitonin family of receptors. Another region of the putative receptor, however, is similar to sequences of serotonin receptors and other receptors of the socalled rhodopsin family of GPCRs. The rhodopsin family has numerous members in higher vertebrate species. Alignments and phylogenetic analyses of the regions of similarity yielded results in accordance with other evolutionary considerations. Our cDNA thus occurred on a distinct major branch in relation to the rest of the rhodopsin family. In relation to the calcitonin family, our cDNA and CAMP receptors occurred together on a distinct major branch but appear to have diverged from each other shortly after their divergence from the rest of the calcitonin family. Other features further argue for a tentative identification of it as a GPCR. It displays seven discrete and strongly predicted transmembrane domains when analyzed in hydropathy plots. The preferred orientation is with the amino terminus towards the outside. It has one Cys residue in extracellular loop 1 and another in extracellular loop 2. Cys residues in these loops are known to form disulfide bridges in many other GPCRs. Finally, it has several fully conserved amino acids that belong to the most conserved in previously known GPCRs, that occur in the above regions of similarity.
Expression of Rat cGMP‐Binding cGMP‐Specific Phosphodiesterase mRNA in Purkinje Cell Layers During Postnatal Neuronal Development
Authors: Kotera Jun , Yanaka Noriyuki , Fujishige Kotomi , Imai Yuji , Akatsuka Hiroyuki , Ishizuka Tohru , Kawashima Keisuke , Omori Kenji
Published: June 2004

The cDNA encoding rat cGMP‐binding, cGMP‐specific phosphodiesterase (cGB‐PDE) was isolated from a rat lung cDNA library. Although the deduced amino acid sequence showed 93.4% similarity with that of bovine cGB‐PDE, the N‐terminal portion of rat cGB‐PDE was extremely different from that of bovine. Northern blot analysis indicated that cGB‐PDE transcripts in rats were expressed not only in aorta and lung, but also in several other tissues including cerebellum. In situ hybridization analysis demonstrated that cerebellar expression of cGB‐PDE was confined to Purkinje cell layers in adult rats. To clarify the role of cGB‐PDE in the cerebellum, we investigated expression of cGB‐PDE mRNA in rats of various ages. cGB‐PDE mRNA was not observed in the cerebellum of newborn rats, but levels of a cGB‐PDE mRNA were markedly increased between 4 days and 28 days of age and reached a maximum in eight‐week‐old rats. In this study, we suggest that cGB‐PDE plays important roles not only in regulating the relaxation of vascular vessels, but also in establishing neuronal networks in the cerebellum at an early postnatal stage. In addition the NO/cGMP/cGB‐PDE pathway appears to be essential for the induction of long‐term depression.
In Vitro Assembly Properties of Purified Bacterially Expressed Capsid Proteins of Human Immunodeficiency Virus
Authors: Gross Ingolf , Hohenberg Heinz , Kräusslich Hans‐Georg
Published: June 2004

The Gag polyprotein of retroviruses is sufficient for assembly and budding of virus‐like particles from the host cell. In the case of human immunodeficiency virus (HIV), Gag contains the domains matrix, capsid (CA), nucleocapsid (NC) and p6 which are separated by the viral proteinase inside the nascent virion, leading to morphological maturation to yield an infectious virus. In the mature virus, CA forms a capsid shell surrounding the ribonucleoprotein core consisting of NC and the genomic RNA. To define requirements for particle assembly and functional contributions of individual domains, we expressed domains of HIV Gag in Escherichia coli and purified the products to near homogeneity. In vitro assembly of CA, with or without the C‐terminally adjacent spacer peptide, yielded tubular structures with a diameter of approximately 55 nm and heterogeneous length. Efficient particle formation required high protein concentration, high salt and neutral to alkaline pH. In contrast, in vitro assembly of CA‐NC occurred at a 20‐fold lower protein concentration and in low salt, but required addition of RNA. These results suggest that hydrophobic interactions of capsid proteins are sufficient for particle formation while the RNA‐binding nucleocapsid domain may concentrate and align structural proteins on the viral genome.
Subcellular Localization, Substrate Specificity and Crystallization of Duodenase, A Potential Activator of Enteropeptidase
Authors: Zamolodchikova Tatyana S. , Sokolova Elena A. , Alexandrov Sergey L. , Mikhaleva Inessa I. , Prudchenko Igor A. , Morozov Igor A. , Kononenko Neonyla V. , Mirgorodskaya Olga A. , Da Ui , Larionova Natalya I. , Pozdnev Vladimir F. , Ghosh Debashis , Duax William L. , Vorotyntseva Tatyana I.
Published: June 2004

Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A‐gold immunocyto‐chemical method. Duodenase exhibits trypsin‐like and chymotrypsin‐like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be α‐N‐tosylglycylprolyllysine 4‐nitroanilide (kcat/Km of 35 000 M−1 s−1), α‐N‐succinylthreonylprolyllysme 4‐nitroanilide (kcat/Km of 18000 M−1 s−1) and α‐N‐serylprolyllysine 4‐nitroanilide (Kcat/km of 2600 M−1 s−1), all of which contain the P1–P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9‐P5'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (Km of 87 μ; kcat of 1.4 s−1; kcat/Km of 16000 M−1 s−1). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron X‐rays to 0.2‐nm resolution.
Signal Transduction in Fibroblasts Stably Transformed by [Val12]Ras
Authors: Ljungdahl Sofia , Linder Stig , Sollerbrant Kerstin , Svensson Catharina , Shoshan Maria C.
Published: June 2004

Ras‐transformed cells often show high levels of expression of activating protein‐1 and Ets and of genes regulated by these transcription factors. In analogy with the effects of transient stimulation of Ras, it is assumed that the increase in transcription‐factor trans activation in stably transformed cells is due to Ras‐induced constitutive activation of mitogen‐activated protein kinases. However, this has not been extensively studied. Using specific substrate peptides, we have examined here the activities of two types of mitogen‐activated protein kinase, extracellular‐signal‐regulated kinase (ERK) and Jun N‐terminal kinase (JNK), in [Val12]Ras‐transformed rat embryo fibroblast cell lines. These activities were elevated 2–3‐fold in Ras‐transformed cells compared with non‐transformed cells with a similar growth rate. Increased ERK activity was not necessarily accompanied by a similar increase in JNK activity. In transformed cells, ERK and JNK activities could be stimulated fourfold and ninefold by phorbol ester and ultraviolet‐light treatment, respectively, indicating that only a fraction of these enzymes were constitutively activated in these cells. It has been suggested that inactive JNK downregulates c‐Jun transcriptional activity by binding to the c‐Jun δ‐domain. No decrease in δ‐inhibitor activity could be demonstrated in Ras‐transformed cells compared with control cells, consistent with the presence of mainly inactive JNK in transformed cells. Treatment of transformed cells with benzodiazepine 5B, an inhibitor of Ras farnesylation, decreased ERK and JNK activities, and concomitantly caused morphological reversion, reduced growth rate, and normalization of transformation‐related gene expression. We conclude that in stably Ras‐transformed cells the moderately increased ERK/JNK activities are not coregulated, and that ERK rather than JNK activity correlated with transformation‐related gene expression.