bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum amyloid A, the major vertebrate acute‐phase reactant
Authors: Uhlar Clarissa M. , Whitehead Alexander S.
Published: March 2003

The serum amyloid A (SAA) family comprises a number of differentially expressed apolipoproteins, acute‐phase SAAs (A‐SAAs) and constitutive SAAs (C‐SAAs). A‐SAAs are major acute‐phase reactants, the in vivo concentrations of which increase by as much as 1000‐fold during inflammation. A‐SAA mRNAs or proteins have been identified in all vertebrates investigated to date and are highly conserved. In contrast, C‐SAAs are induced minimally, if at all, during the acute‐phase response and have only been found in human and mouse. Although the liver is the primary site of synthesis of both A‐SAA and C‐SAA, extrahepatic production has been reported for most family members in most of the mammalian species studied. In vitro, the dramatic induction of A‐SAA mRNA in response to pro‐inflammatory stimuli is due largely to the synergistic effects of cytokine signaling pathways, principally those of the interleukin‐1 and interleukin‐6 type cytokines. This induction can be enhanced by glucocorticoids. Studies of the A‐SAA promoters in several mammalian species have identified a range of transcription factors that are variously involved in defining both cytokine responsiveness and cell specificity. These include NF‐κB, C/EBP, YY1, AP‐2, SAF and Sp1. A‐SAA is also post‐transcriptionally regulated. Although the precise role of A‐SAA in host defense during inflammation has not been defined, many potential clinically important functions have been proposed for individual SAA family members. These include involvement in lipid metabolism/transport, induction of extracellular‐matrix‐degrading enzymes, and chemotactic recruitment of inflammatory cells to sites of inflammation. A‐SAA is potentially involved in the pathogenesis of several chronic inflammatory diseases: it is the precursor of the amyloid A protein deposited in amyloid A amyloidosis, and it has also been implicated in the pathogenesis of atheroscelerosis and rheumatoid arthritis.
Unfolding studies of human adenovirus type 2 fibre trimers
Authors: Mitraki Anna , Barge Annie , Chroboczek Jadwiga , Andrieu Jean‐Pierre , Gagnon Jean , Ruigrok Rob W. H.
Published: March 2003

Adenovirus fibres are trimeric proteins that protrude from the 12 fivefold vertices of the virion and are the cell attachment organelle of the virus. They consist of three segments: an N‐terminal tail, which is noncovalently attached to the penton base, a thin shaft carrying 15 amino acid pseudo repeats, and a C‐terminal globular head (or knob) which recognizes the primary cell receptor. Due to their exceptional stability, which allows easy distinction of native trimers from unfolded forms and folding intermediates, adenovirus fibres are a very good model system for studying folding in vivo and in vitro. To understand the folding and stability of the trimeric fibres, the unfolding pathway of adenovirus 2 fibres induced by SDS and temperature has been investigated. Unfolding starts from the N‐terminus and a stable intermediate accumulates that has the C‐terminal head and part of the shaft structure (shown by electron microscopy). The unfolded part can be digested away using limited proteolysis, and the precise digestion sites have been determined. The remaining structured fragment is recognized by monoclonal antibodies that are specific for the trimeric globular head and therefore retains a native trimeric structure. Taken together, our results indicate that adenovirus fibres carry a stable C‐terminal domain, consisting of the knob with five shaft‐repeats.
Cloning and over‐expression in Escherichia coli of the gene encoding NADPH group III alcohol dehydrogenase from Thermococcus hydrothermalis
Authors: Antoine Elisabeth , Rolland Jean‐Luc , Raffin Jean‐Paul , Dietrich Jacques
Published: March 2003

A NADP‐dependent group III alcohol dehydrogenase (ADH) was purified from the hyperthermophilic strictly anaerobic archaeon Thermococcus hydrothermalis, which grows at an optimum temperature of 85 °C and an optimum pH of 6. The gene encoding this enzyme was cloned, sequenced, and over‐expressed in Escherichia coli. The recombinant enzyme was purified, characterized and compared with the native form of the enzyme. The enzyme structure is pH‐dependent, being a 197‐kDa tetramer (subunit of 45 kDa) at pH 10.5, the pH optimum for alcohol oxidation, and a 80.5‐kDa dimer at pH 7.5, the pH optimum for aldehyde reduction. The kinetic parameters of the enzyme show that the affinity of the enzyme is greater for the aldehyde substrate and NADPH cofactor, suggesting that the dimeric form of the enzyme is probably the active form in vivo. The ADH of T. hydrothermalis oxidizes a series of primary aliphatic and aromatic alcohols preferentially from C2 to C8 but is also active towards methanol and glycerol and stereospecific for monoterpenes. T. hydrothermalis ADH is the first Thermococcale ADH to be cloned and overproduced in a mesophilic heterologous expression system, and the recombinant and the native forms have identical main characteristics.
Purification and properties of a basic endo‐β‐1,6‐glucanase (BGN16.1) from the antagonistic fungus Trichoderma harzianum
Authors: de la Cruz Jesús , Llobell Antonio
Published: March 2003

The antagonistic fungus Trichoderma harzianum CECT 2413 produces at least two extracellular β‐1,6‐glucanases, among other hydrolases acting on polysaccharides from fungal cell walls, when grown in chitin as the sole carbon source. We have previously reported on the purification and biochemical characterization of the major activity, which corresponds to an acidic enzyme named BGN16.2 [de la Cruz, J., Pintor‐Toro, J.A., Benítez, T. & Llobell, A. (1995) J. Bacteriol.177, 1864–1871]. In this paper, we report on the purification to electrophoretical homogeneity of BGN16.1, the second β‐1,6‐glucanase enzyme. BGN16.1 was purified by ammonium sulfate precipitation followed by adsorption and digestion of pustulan (a β‐1,6‐glucan), chromatofocusing and gel‐filtration chromatography. BGN16.1 is a non‐glycosylated protein with an apparent molecular mass of 51 kDa and a basic isoelectric point (pI 7.4–7.7). The enzyme was active toward substrates containing β‐1,6‐glycosidic linkages, including yeast cell walls. The Km was 0.8 mg·mL−1 with pustulan as the substrate. Reaction product analysis by HPLC clearly indicated that BGN16.1 has an endo‐hydrolytic mode of action. The probable role of this enzyme in the antagonistic action of T. harzianum is also discussed.
Polymorphism of turnip yellow mosaic virus empty shells and evidence for conformational changes occurring after release of the viral RNA
Authors: Michels Bernard , Leimkühler Martina , Lechner M. Dieter , Adrian Marc , Lorber Bernard , Witz Jean
Published: March 2003

Turnip yellow mosaic virus (TYMV) is a small isometric plant virus which decapsidates by releasing its RNA through a hole in the capsid, leaving behind an empty shell [R. E. F. Matthews and J. Witz, (1985) Virology144, 318–327]. Similar empty shells (artificial top component, ATC) can be obtained by submitting the virions to various treatments in vitro. We have used differential scanning calorimetry, analytical sedimentation, and electron microscopy to investigate the thermodenaturation of natural empty shells (NTC, natural top component) present in purified virus suspensions, and of several types of ATCs. ATCs divided in two major classes. Those obtained by alkaline titration, by the action of urea or butanol behaved as NTC: their thermograms contained only one peak corresponding to the irreversible dissociation of the shells and the denaturation of the coat protein. The temperature of this unique transition varied significantly with pH, from 71 °C at pH 4.5 to 84 °C at pH 8.5. The thermograms of ATCs obtained by freezing and thawing, or by the action of high pressure, contained two peaks: shells dissociated first into smaller protein aggregates at 57 °C (at pH 5.0) to 61 °C (at pH 8.5), which denatured at the temperature of the unique transition of NTC. Shells obtained by heating virions to 55 °C at pH 7.6, changed conformation after the release of the viral RNA, as upon continuous heating to 95 °C, their thermograms were similar to those of the shells obtained by freezing and thawing, whereas after purification they behaved like NTC. Structural implications of these observations are discussed.
Translocation between membranes and cytosol of p42IP4, a specific inositol 1,3,4,5‐tetrakisphosphate/phosphatidylinositol 3,4,5‐trisphosphate‐receptor protein from brain, is induced by inositol 1,3,4,5‐tetrakisphosphate and regulated by a membrane‐associated 5‐phosphatase
Authors: Stricker Rolf , Adelt Stephan , Vogel Günter , Reiser Georg
Published: March 2003

The highly conserved 42‐kDa protein, p42IP4 was identified recently from porcine brain. It has also been identified similarly in bovine, rat and human brain as a protein with two pleckstrin homology domains that binds Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3 with high affinity and selectivity. The brain‐specific p42IP4 occurs both as membrane‐associated and cytosolic protein. Here, we investigate whether p42IP4 can be translocated from membranes by ligand interaction. p42IP4 is released from cerebellar membranes by incubation with Ins(1,3,4,5)P4. This dissociation is concentration‐dependent (> 100 nm), occurs within a few minutes and and is ligand‐specific. p42IP4 specifically associates with PtdIns(3,4,5)P3‐containing lipid vesicles and can dissociate from these vesicles by addition of Ins(1,3,4,5)P4. p42IP4 is only transiently translocated from the membranes as Ins(1,3,4,5)P4 can be degraded by a membrane‐associated 5‐phosphatase to Ins(1,3,4)P3. Then, p42IP4 re‐binds to the membranes from which it can be re‐released by re‐addition of Ins(1,3,4,5)P4. Thus, Ins(1,3,4,5)P4 specifically induces the dissociation from membranes of a PtdIns(3,4,5)P3 binding protein that can reversibly re‐associate with the membranes. Quantitative analysis of the inositol phosphates in rat brain tissue revealed a concentration of Ins(1,3,4,5)P4 comparable to that required for p42IP4 translocation. Thus, in vivo p42IP4 might interact with membranes in a ligand‐controlled manner and be involved in physiological processes induced by the two second messengers Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3.
Expression, partial purification and functional properties of themuscle‐specific calpain isoform p94
Authors: Branca Donata , Gugliucci Arianna , Bano Daniele , Brini Marisa , Carafoli Ernesto
Published: March 2003

The muscle‐specific calpain isoform p94 has high propensity to autocatalytic degradation, thus no significant amounts of the intact active protein have been available so far. As a result, aspects like its regulation (via Ca2+ and other factors) and its intracellular localization are unknown or obscure. In this work, large amounts of human p94 have been produced in insect cells using a recombinant baculovirus expression system. Although most of the protease was recovered in an insoluble and catalytically inactive form, the soluble fraction contained amounts of intact active p94 adequate for its characterization. His‐tagged recombinant p94, obtained by the same expression system, was partially purified as an active product. Both the unmodified and the partially purified His‐tagged p94 bound calcium with high affinity, and their autolytic activity required Ca2+. The sensitivity of the catalytic activity of the recombinant protease to Ca2+ was very high. In fact, p94 in soluble cell extracts autolysed to a significant extent even in the presence of submicromolar Ca2+ levels. Thus, in analogy to what demonstrated for the ubiquitous m‐ and µ‐calpain isoforms, intracellular Ca2+ might be one of the factors controlling the activity of this muscle‐specific calpain isoform.
Localization of cyanobacterial photosystem II donor‐side subunits by electron microscopy and the supramolecular organization ofphotosystem II in the thylakoid membrane
Authors: Kuhl Helena , Rögner Matthias , van Breemen Jan F. L. , Boekema Egbert J.
Published: March 2003

A large set of electron microscopy projections of photosystem II (PSII) dimers isolated from the cyanobacterium Synechococcus elongatus was characterized by single particle image analysis. In addition to previously published maps at lower resolution [Boekema, E.J., Hankamer, B., Bald, D., Kruip, J., Nield, J., Boonstra, A.F., Barber, J. & Rögner, M. (1995) Proc. Natl Acad. Sci. USA92, 175–179], the new side‐view projections show densities of all three lumenal extrinsic proteins, i.e. the 33‐kDa, 12‐kDa and the cytochrome c‐550 subunit encoded by psbO, psbU and psbV, respectively. Analysis of the size and shape of the top‐view projections revealed a small number of photosystem II particles of about double the size of the usual dimers. Size and quantity of these ‘double dimers’ correlates with a small fraction of 1000‐kDa particles found with HPLC‐size‐exclusion chromatographic analysis. Because many cyanobacteria contain dimeric photosystem II complexes arranged in rows within the membrane, the double dimers can be considered as the breakdown fragments of these rows. Their analysis enabled the detection of the arrangement of photosystem II within the rows, in which the dimers interact with other dimers mostly with their tips, leaving a rather open center at the interfaces of two dimers. The dimers have a repeating distance of only 11.7 nm. As a consequence, the phycobilisomes, located on top of PSII and functioning in light‐harvesting, must be closely packed or almost touch each other, in a manner similar to a recently suggested model [Bald, D., Kruip, J. & Rögner, M. (1996) Photosynthesis Res.49, 103–118].
Abundant secretory lipocalins displaying male and lactation‐specific expression in adult hamster submandibular gland
Authors: Thavathiru Elangovan , Jana Nihar R. , De Prabir K.
Published: March 2003

We have previously identified massively expressed 24‐ and 20.5‐kDa male‐specific proteins in submandibular salivary glands (SMG) of adult hamsters. Here we report the cloning of the cDNA encoding the 24‐kDa protein which we have now found to be a heterogenously N‐glycosylated form of the 20.5‐kDa protein. The deduced amino acid sequence indicated that the protein is a member of the lipocalin family, the two most related lipocalins being rat odorant‐binding protein of nasal mucosa and aphrodisin, a pheromonal protein present in vaginal discharge and saliva of female hamsters. Northern blot analysis showed that cognate mRNA is expressed in hamster SMG and lacrimal gland (LG) displaying marked sex‐hormonal repression. The sex‐hormonal repression patterns showed similarities and dissimilarities between SMG and LG but they were, respectively, similar to the sex‐hormonal repression pattern noted for the SMG 24/20.5‐kDa male‐specific proteins and for an abundant female‐specific 20‐kDa LG secretory protein. These SMG and LG proteins were found to be immunologically similar and secretion of the SMG proteins in saliva and their excretion in urine was detected. The male‐specific and abundant expression of the SMG proteins were seen at and after sexual maturity but was not dependent on androgens. Surprisingly, a temporary male‐like expression of these SMG proteins was seen in lactating females which was obliterated by oestrogen administration. Our results show that despite differences in their repression by sex hormones, the gene for SMG 24/20.5‐kDa proteins is similar or identical to that of LG 20‐kDa protein and their marked repression by both androgens and oestrogens might be at the transcriptional level. Moreover, they might be excellent models with which to study sex hormone repression of gene expression at the molecular level. The results of homology search and the male‐ and lactation‐specific pressure of the SMG proteins in adult saliva and urine suggests a possibility of their involvement in olfaction‐mediated chemical communication between hamsters.
The prothrombin gene is expressed in the rat kidney
Authors: Grover Phulwinder K. , Dogra Satish C. , Davidson Benjamin P. , Stapleton Alan M. F. , Ryall Rosemary L.
Published: March 2003

There is considerable interest in determining the role of prothrombin fragments, especially urinary prothrombin fragment 1 (UPTF1), in the pathogenesis of calcium oxalate (CaOx) urinary calculi. This fragment is present in abundance in the matrix of CaOx crystals generated in human urine in vitro and has also been detected in human urinary stones containing calcium. More recently, prothrombin gene expression has been reported in the human kidney. However, studies examining the renal biosynthesis of prothrombin or perhaps only its fragments during experimental lithogenesis, and in consequence, the role of UPTF1 in stone formation, cannot be carried out in humans. The aim of this investigation therefore was to determine whether prothrombin gene expression is present in the rat kidney.

Total RNA was isolated from the kidneys and livers of 12 rats. Using reverse transcriptase PCR, mRNAs corresponding to the thrombin and fragment 1 + 2 (F1+2) regions of prothrombin were analysed by agarose gel electrophoresis. The expression of glyceraldehyde 3‐phosphate dehydrogenase was also examined to determine whether the quality of the tissue mRNAs was adequate for analyses. The amplified products were identified by sequence analysis.

All kidneys displayed evidence of expression of the thrombin and F1+2 domains of the prothrombin gene. Furthermore, the sequences of these PCR‐derived products from kidney were identical to those from liver. This suggests that the prothrombins secreted by these two organs are identical. The fact that prothrombin biosynthesis occurs in both the human and rat kidney presents an opportunity for using established rat models of stone disease to evaluate the influence of lithogenic conditions on prothrombin gene expression, and the potential role of UPTF1 in vivo.
Lipopolysaccharide structures of Helicobacter pylori genomic strains 26695 and J99, mouse model H. pylori Sydney strain, H. pylori P466 carrying sialyl Lewis X, and H. pylori UA915 expressing Lewis B
Authors: Monteiro Mario A. , Appelmelk Ben J. , Rasko David A. , Moran Anthony P. , Hynes Sean O. , MacLean Leann L. , Chan Ken H. , Michael Frank St , Logan Susan M. , O'Rourke Jani , Lee Adrian , Taylor Diane E. , Perry Malcolm B.
Published: March 2003

This study describes the molecular makeup of the cell‐wall lipopolysaccharides (LPSs) (O‐chain polysaccharide→core oligosaccharide→lipid A) from five Helicobacter pylori strains: H. pylori 26695 and J99, the complete genome sequences of which have been published, the established mouse model Sydney strain (SS1), and the symptomatic strains P466 and UA915. All chemical and serological experiments were performed on the intact LPSs. H. pylori 26695 and SS1 possessed either a low‐Mr semi‐rough‐form LPS carrying mostly a single Ley type‐2 blood‐group determinant in the O‐chain region covalently attached to the core oligosaccharide or a high‐Mr smooth‐form LPS, as did strain J99, with an elongated partially fucosylated type‐2 N‐acetyllactosamine (polyLacNAc) O‐chain polymer, terminated mainly by a Lex blood‐group determinant, connected to the core oligosaccharide. In the midst of semi‐rough‐form LPS glycoforms, H. pylori 26695 and SS1 also expressed in the O‐chain region a difucosylated antigen, α‐l‐Fucp(1–3)‐α‐l‐Fucp(1–4)‐β‐d‐GlcpNAc, and the cancer‐cell‐related type‐1 or type‐2 linear B‐blood‐group antigen, α‐d‐Galp(1–3)‐β‐d‐Galp(1–3 or 4)‐β‐d‐GlcpNAc. The LPS of H. pylori strain P466 carried the cancer‐associated type‐2 sialyl Lex blood‐group antigen, and the LPS from strain UA915 expressed a type‐1 Leb blood‐group unit. These findings should aid investigations that focus on identifying and characterizing genes responsible for LPS biosynthesis in genomic strains 26695 and J99, and in understanding the role of H. pylori LPS in animal model studies. The LPSs from the H. pylori strains studied to date were grouped into specific glycotype families.
Analysis of the tangled relationships between P‐glycoprotein‐mediated multidrug resistance and the lipid phase of the cell membrane
Authors: Ferté Jacques
Published: March 2003

P‐glycoprotein (Pgp), the so‐called multidrug transporter, is a plasma membrane glycoprotein often involved in the resistance of cancer cells towards multiple anticancer agents in the multidrug‐resistant (MDR) phenotype. It has long been recognized that the lipid phase of the plasma membrane plays an important role with respect to multidrug resistance and Pgp because: the compounds involved in the MDR phenotype are hydrophobic and diffuse passively through the membrane; Pgp domains involved in drug binding are located within the putative transmembrane segments; Pgp activity is highly sensitive to its lipid environment; and Pgp may be involved in lipid trafficking and metabolism. Unraveling the different roles played by the membrane lipid phase in MDR is relevant, not only to the evaluation of the precise role of Pgp, but also to the understanding of the mechanism of action and function of Pgp. With this aim, I review the data from different fields (cancer research, medicinal chemistry, membrane biophysics, pharmaceutical research) concerning drug–membrane, as well as Pgp–membrane, interactions. It is emphasized that the lipid phase of the membrane cannot be overlooked while investigating the MDR phenotype. Taking into account these aspects should be useful in the search of ways to obviate MDR and could also be relevant to the study of other multidrug transporters.
Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs Rana luteiventris, Rana berlandieri and Rana pipiens
Authors: Goraya Jadvinder , Wang Yuqi , Li Zhihong , O'Flaherty Martina , Knoop Floyd C. , Platz James E. , Conlon J. Michael
Published: March 2003

The skins of frogs of the genus Rana synthesize a complex array of antimicrobial peptides that may be grouped into eight families on the basis of structural similarity. A total of 24 peptides with differential growth‐inhibitory activity towards the Gram‐positive bacterium Staphylococcus aureus, the Gram‐negative bacterium Escherichia coli and the yeast Candida albicans were isolated from extracts of the skins of three closely related North American frogs, Rana luteiventris (spotted frog), Rana berlandieri (Rio Grande leopard frog) and Rana pipiens (Northern leopard frog). Structural characterization of the antimicrobial peptides demonstrated that they belonged to four of the known families: the brevinin‐1 family, first identified in skin of the Asian frog Rana porosa brevipoda; the esculentin‐2 family, first identified in the European frog Rana esculenta; the ranatuerin‐2 family, first identified in the North American bullfrog Rana catesbeiana; and the temporin family, first identified in the European frog Rana temporaria. Peptides belonging to the brevinin‐2, ranalexin, esculentin‐1 and ranatuerin‐1 families were not identified in the extracts. Despite the close phylogenetic relationship between the various species of Ranid frogs, the distribution and amino‐acid sequences of the antimicrobial peptides produced by each species are highly variable and species‐specific, suggesting that they may be valuable in taxonomic classification and molecular phylogenetic analysis.
Molecular characterization of a human scavenger receptor, human MARCO
Authors: Elshourbagy Nabil A. , Li Xiaotong , Terrett John , VanHorn Stephanie , Gross Mitchell S. , Adamou John E. , Anderson Karen M. , Webb Christine L. , Lysko Paul G.
Published: March 2003

Murine MARCO has been identified recently in subsets of macrophages located in the peritoneum, marginal zone of the spleen, and the medullary cord of lymph nodes, where it has been proposed that it serves as a bacteria‐binding receptor. A scavenger receptor family member with an extended collagenous domain, murine MARCO has also been demonstrated in atherosclerotic lesions of susceptible mice. We report here the identification, tissue and chromosomal localization, and pharmacological characterization of human (h)MARCO. hMARCO was identified from a macrophage cDNA library by electronic screening with the murine MARCO sequence. Nucleotide sequence analysis confirmed that the full‐length hMARCO clone encoded a 519‐amino acid protein sharing 68.5% identity with murine MARCO. RNA blot analysis indicated that the hMARCO transcript is 2.0 kb in length and is predominantly expressed in human lung, liver, and lymph nodes. Radiation hybrid mapping localized hMARCO to chromosome 2q14. Ligand‐binding studies of COS cells expressing hMARCO demonstrated significant specific binding of both Escherichia coli and Staphylococcus aureus. In contrast, the hMARCO receptor expressed in COS cells did not specifically bind the scavenger receptor ligand acetylated low‐density lipoprotein (LDL), despite its similarity to the elongated collagen‐like binding domain of the macrophage scavenger receptor. In addition, acetylated (Ac)LDL and oxidized (Ox)LDL did not inhibit E. coli binding to hMARCO. These data suggest that hMARCO may play an important role in host defense, but it has no obvious role in the accumulation of modified lipoproteins during atherogenesis.
Maculatin 1.1, an anti‐microbial peptide from the Australian tree frog, Litoria genimaculata
Authors: Chia Brian C. S. , Carver John A. , Mulhern Terrence D. , Bowie John H.
Published: March 2003

The dorsal glands of Australian tree frogs from the Litoria species contain a diversity of antibiotic peptides that forms part of the defence system of the animal. Here, the antibiotic activity and structure of maculatin 1.1, a 21 amino acid peptide from Litoria genimaculata, are compared. The activity data on maculatin 1.1 and a series of its analogues imply that the mechanism of action of maculatin 1.1 involves binding to, and subsequent lysis of, the bacterial cell membrane. The structure of maculatin 1.1 was determined using NMR spectroscopy in a trifluoroethanol/water mixture and when incorporated into dodecylphosphocholine micelles. Under both conditions, the peptide adopts a very similar conformation, i.e. a helical structure with a central kink in the vicinity of Pro15. The kink allows the peptide to adopt a well‐defined amphipathic conformation along its entire length. The similar structures determined under both solvent conditions imply that structures of membrane‐interacting peptides in trifluoroethanol/water mixtures are representative of those adopted in a membrane environment, e.g. when incorporated into micelles. The synthetic Ala15 analogue of maculatin 1.1 has markedly reduced activity and its NMR‐derived structure is a well‐defined helix, which lacks the central kink and flexibility of the parent molecule. It is concluded that the kink is important for full biological activity of the peptide, probably because it allows maximum amphipathicity of the peptide to facilitate interaction with the membrane. The structure of maculatin 1.1 is compared with a related peptide, caerin 1.1 [Wong, H., Bowie, J.H. and Carver, J.A. (1997) Eur. J. Biochem. 247, 545–557], which has an additional central proline residue and enhanced central flexibility compared with maculatin 1.1. The role of central flexibility within antibiotic peptides in their interaction with bacterial membranes is discussed.
Stabilization of NAD‐dependent formate dehydrogenase from Candida boidinii by site‐directed mutagenesis of cysteine residues
Authors: Slusarczyk Heike , Felber Stephan , Kula Maria‐Regina , Pohl Martina
Published: March 2003

The gene of the NAD‐dependent formate dehydrogenase (FDH) from the yeast Candida boidinii was cloned by PCR using genomic DNA as a template. Expression of the gene in Escherichia coli yielded functional FDH with about 20% of the soluble cell protein. To confirm the hypothesis of a thiol‐coupled inactivation process, both cysteine residues in the primary structure of the enzyme have been exchanged by site‐directed mutagenesis using a homology model based on the 3D structure of FDH from Pseudomonas sp. 101 and from related dehydrogenases. Compared to the wt enzyme, most of the mutants were significantly more stable towards oxidative stress in the presence of Cu(II) ions, whereas the temperature optima and kinetic constants of the enzymatic reaction are not significantly altered by the mutations. Determination of the Tm values revealed that the stability at temperatures above 50 °C is optimal for the native and the recombinant wt enzyme (Tm 57 °C), whereas the Tm values of the mutant enzymes vary in the range 44–52 °C. Best results in initial tests concerning the application of the enzyme for regeneration of NADH in biotransformation of trimethyl pyruvate to ltert leucine were obtained with two mutants, FDHC23S and FDHC23S/C262A, which are significantly more stable than the wt enzyme.
DNase I hypersensitive sites and transcriptional activation of the lamin A/C gene
Authors: Nakamachi Kazuhiko , Nakajima Noboru
Published: March 2003

The lamin A/C gene encodes subtypes of nuclear lamins, which are involved in nuclear envelope formation, and was recently identified as the responsible gene for the autosomal dominant Emery–Dreifuss muscular dystrophy. Expression of the lamin A/C gene is developmentally regulated but little is known about the regulatory mechanism. Previous studies of lamin A/C expression suggested that the chromatin structure is important for the regulation of its expression. To elucidate the regulatory mechanism of the lamin A/C gene expression, we have analysed the functional region of the mouse lamin A/C promoter and the chromatin structure of the gene in terms of nucleosome structure and DNase I hypersensitivity. Our analyses revealed disruption of the nucleosome array at the promoter region and the presence of multiple DNase I hypersensitive sites (HSs) which were specifically associated with expression of the lamin A/C gene. Inclusion of a segment which contained the HSs in a lamin A/C promoter‐luciferase reporter plasmid showed no effect on the transfected promoter activity in transient expression assays. On the other hand, substantial enhancement of the promoter activity was detected when the transfected DNA was stably integrated into the genome, suggesting the importance of the HSs in the regulation of lamin A/C expression.
Assessment of amino‐acid substitutions at tryptophan 16 in α‐galactosidase
Authors: Maranville Elizabeth , Zhu Alex
Published: March 2003

The tryptophan residue at position 16 of coffee bean α‐galactosidase has previously been shown to be essential for enzyme activity. The potential role of this residue in the catalytic mechanism has been further studied by using site‐directed mutagenesis to substitute every other amino acid for tryptophan at that site. Mutant enzymes were expressed in Pichia pastoris, a methylotrophic yeast strain, and their kinetic parameters were calculated. Only amino acids containing aromatic rings (phenylalanine and tyrosine) were able to support a significant amount of enzyme activity, but the kinetics and pH profiles of these mutants differed from wild‐type. Substitution of arginine, lysine, methionine, or cysteine at position 16 allowed a small amount of enzyme activity with the optimal pH shifted towards more acidic. All other residues abolished enzyme activity. Our data support the hypothesis that tryptophan 16 is affecting the pKa of a carboxyl group at the active site that participates in catalysis. We also describe an assay for continuously measuring enzyme kinetics using fluorogenic 4‐methylumbelliferyl substrates. This is useful in screening enzymes from colonies and determining the enzyme kinetics when the enzyme concentration is not known.
Several dinucleoside polyphosphates are acceptor substrates in the T4 RNA ligase catalyzed reaction
Authors: Atencia Eva A. , Montes María , Günther Sillero María A. , Sillero Antonio
Published: March 2003

Several dinucleoside polyphosphates accept cytidine‐3′,5′‐bisphosphate from the adenylylated donor 5′‐adenylylated cytidine 5′,3′‐bisphosphate in the T4 RNA ligase catalyzed reaction. The 5′‐adenylylated cytidine 5′,3′‐bisphosphate synthesized in a first step, from ATP and cytidine‐3′,5′‐bisphosphate, is used as a substrate to transfer the cytidine‐3′,5′‐bisphosphate residue to the 3′‐OH group(s) of diguanosine tetraphosphate (Gp4G) giving rise to Gp4GpCp and pCpGp4GpCp in a ratio of ≈ 10 : 1, respectively. The synthesized Gp4GpCp was characterized by treatment with snake venom phosphodiesterase and alkaline phosphatase and analysis (chromatographic position and UV spectra) of the reaction products by HPLC. The apparent Km values measured for Gp4G and 5′‐adenylylated cytidine 5′,3′‐bisphosphate in this reaction were ≈ 4 mm and 0.4 mm, respectively. In the presence of 0.5 mm ATP and 0.5 mm cytidine‐3′,5′‐bisphosphate, the relative efficiencies of the following nucleoside(5′)oligophospho(5′)nucleosides as acceptors of cytidine‐3′,5′‐bisphosphate from 5′‐adenylylated cytidine 5′,3′‐bisphosphate are indicated in parentheses: Gp4G (100); Gp5G (101); Ap4G (47); Ap4A (39). Gp2G, Gp3G and Xp4X were not substrates of the reaction. Dinucleotides containing two guanines and at least four inner phosphates were the preferred acceptors of cytidine‐3′,5′‐bisphosphate at their 3′‐OH group(s).
Quantitative analysis of gene expression with an improved green fluorescent protein
Authors: Scholz Oliver , Thiel Anja , Hillen Wolfgang , Niederweis Michael
Published: March 2003

The fast and easy in vivo detection predestines the green fluorescent protein (GFP) for its use as a reporter to quantify promoter activities. We have increased the sensitivity of GFP detection 320‐fold compared to the wild‐type by constructing gfp+, which contains mutations improving the folding efficiency and the fluorescence yield of GFP+. Twelve expression levels were measured using fusions of the gfp+ and lacZ genes with the tetA promoter in Escherichia coli. The agreement of GFP+ fluorescence with β‐galactosidase activities was excellent, demonstrating that the gfp+ gene can be used to accurately quantify gene expression in vivo. However, expression of the gfp+ gene from the stronger hsp60 promoter revealed that high cellular concentrations of GFP+ caused an inner filter effect reducing the fluorescence by 50%, thus underestimating promoter activity. This effect is probably due to the higher absorbance of cells containing GFP+. Thus promoters with activities differing by about two orders of magnitude can be correctly quantified using the gfp+ gene. Possibilities of using GFP variants beyond this range are discussed.
Salmon antithrombin has only three carbohydrate side chains, and shows functional similarities to human β‐antithrombin
Authors: Andersen Øivind , Flengsrud Ragnar , Norberg Kari , Salte Ragnar
Published: March 2003

Antithrombin, a major coagulation inhibitor in mammals, has for the first time been cDNA cloned from a fish species. The predicted mature liver antithrombin of Atlantic salmon (Salmo salar) consists of 430 amino acids and shows about 67% sequence identity to mammalian and chicken antithrombins. Due to a single nucleotide replacement, Asn135 of the antithrombin in higher vertebrates is substituted by Asp in the salmon homolog. Hence, in contrast to the vertebrate antithrombins known so far, salmon antithrombin lacks the potential glycosylation site located close to the heparin binding site. The existence of only three N‐linked side chains is evidenced by the sequential removal of three carbohydrate chains from salmon antithrombin during timed‐digestion with N‐glycosidase F. The high heparin binding affinity of the salmon inhibitor, Kd of 2.2 and 48 nm at I = 0.15 and 0.3, respectively, is very similar to that of the minor human isoform β‐antithrombin, which is not glycosylated at Asn135. Furthermore, the invariant third‐position Ser137 at this glycosylation site of mammalian and chicken antithrombins is substituted by Thr in the salmon, a replacement that has been shown to induce full glycosylation in human antithrombin. Thus a rapidly reacting pool of antithrombin may have evolved in two different ways: absence of a glycosylation site in lower vertebrates vs. incomplete glycosylation of a part of the circulating antithrombin in higher vertebrates. Salmon antithrombin appears to have three complex oligosaccharide side chains containing sialic acid terminally linked α(2–3) to galactose, while trace amounts of Galβ(1–4)GlcNAc suggest microheterogeneity due to partial loss of sialic acid.
Activity of wheat α‐amylase inhibitors towards bruchid α‐amylases and structural explanation of observed specificities
Authors: Franco Octávio L. , Rigden Daniel J. , R. Melo Francislete , Bloch Carlos , Silva Carlos P. , Grossi de Sá Maria F.
Published: March 2003

Plant α‐amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Five α‐amylase inhibitors of the structural 0.19 family were isolated from wheat kernels, and assayed against three insect α‐amylases and porcine pancreatic α‐amylase, revealing several intriguing differences in inhibition profiles, even between proteins sharing sequence identity of up to 98%. Inhibition of the enzyme from a commercially important pest, the bean weevil Acanthoscelides obtectus, is observed for the first time. Using the crystal structure of an insect α‐amylase in complex with a structurally related inhibitor, models were constructed and refined of insect and human α‐amylases bound to 0.19 inhibitor. Four key questions posed by the differences in biochemical behaviour between the five inhibitors were successfully explained using these models. Residue size and charge, loop lengths, and the conformational effects of a Cys to Pro mutation, were among the factors responsible for observed differences in specificity. The improved structural understanding of the bases for the 0.19 structural family inhibitor specificity reported here may prove useful in the future for the rational design of inhibitors possessing altered inhibition characteristics.
‘Slave’ metabolites and enzymes
Authors: Teusink Bas , Westerhoff Hans V
Published: March 2003

Although control of fluxes and concentrations tends to be distributed rather than confined to a single rate‐limiting enzyme, the extent of control can differ widely between enzymes in a metabolic network. In some cases, there are enzymes that lack control completely. This paper identifies one surprising origin of such lack of control: If, in a metabolic system, there is a metabolite that affects the catalytic rate of only one enzyme, the corresponding enzyme cannot control any metabolic variable other than the concentration of that metabolite. We call such enzymes ‘slave enzymes’, and the corresponding metabolites ‘slave metabolites’. Implications of the existence of slave enzymes for the control properties of enzymes further down the metabolic pathway are discussed and examined for the glycolytic pathway of yeast. Inadvertent assumptions in metabolic models may cause the latter incorrectly to calculate absence of metabolic control. The phenomenon of slave enzymes may well be important in enhancing metabolic signal transduction.
Self‐assembly and cross‐linking of Volvox extracellular matrix glycoproteins are specifically inhibited by Ellman's reagent
Authors: Sumper Manfred , Nink Jörg , Wenzl Stephan
Published: March 2003

A major impediment to the biochemical characterization of extracellular matrices from algae (as well as higher plants) is the extensive covalent cross‐linking that exists in the matrix, rendering most components insoluble and resistant to conventional extraction procedures. In the multicellular green alga Volvox, biogenesis of the extracellular matrix (ECM) is initiated immediately after the process of embryonic inversion. At this stage of development, the sulfhydryl reagent 5,5′‐dithio‐bis(2‐nitrobenzoic acid), known as Ellman's reagent, interferes in a highly specific manner with ECM biogenesis. Treated post‐inversion embryos are no longer able to assemble an intact ECM and consequently dissociate into a suspension of single cells. Dissociated cells remain viable and continue to secrete ECM proteins into the growth medium, as documented by the identification of several members of the pherophorin family. Cross‐linked ECM polymers such as sulfated surface glycoprotein 185 remain in a soluble state. Thus, treatment with Ellman's reagent opens a simple approach for the isolation and characterization of otherwise inaccessible monomeric precursors.
Structural identification of the lipopolysaccharide O‐antigen produced by Yersinia enterocolitica serotype O:28
Authors: Perry Malcolm B. , MacLean Leann L.
Published: March 2003

The structure of the O‐antigenic polysaccharide (O‐PS) component of the lipopolysaccharide produced by Yersinia enterocolitica serotype O:28 has been elucidated. From chemical methods involving glycose analysis, periodate oxidation, methylation and the use of one‐ and two‐dimensional NMR spectroscopy, the O‐PS was found to be a polymer of repeating branched hexasaccharide units composed of l‐rhamnose (four parts), 2‐acetamido‐2‐deoxy‐d‐glucose (one part), and 2‐acetamido‐2‐deoxy‐d‐galacturonic acid (one part) having the following structure:
UV‐B radiation induced exchange of the D1 reaction centre subunits produced from thepsbA2andpsbA3genes in the cyanobacteriumSynechocystissp. PCC 6803
Authors: Vass Imre , Kirilovsky Diana , Perewoska Irene , Máté Zoltán , Nagy Ferenc , Etienne Anne‐Lise
Published: March 2003

UV‐B irradiation ofSynechocystis6803 cells inhibits photosystem II activity, which can be restored viade novosynthesis of the D1 (and D2) reaction center subunits. Recently we have shown that of the twopsbAgenes that encode identical D1 proteins inSynechocystis6803, UV‐B preferentially enhances the transcription ofpsbA3compared to that ofpsbA2[Máté, Z., Sass, L., Szekeres, M., Vass, I. and Nagy, F. (1998)J. Biol. Chem.273, 17439–17444]. Here we studied the effect of UV‐B on the synthesis of the D1 protein from thepsbA2andpsbA3genes in the P7 mutant ofSynechocystis6803. In this mutant,psbA2carries the Ala251→Val point mutation, which confers resistance to the photosystem II electron transport inhibitor metribuzin, butpsbA3is the same as in the wild‐type. By applying variable chlorophyll fluorescence measurements to distinguish between metribuzin‐sensitive and metribuzin‐resistant photosystem II centers we quantified the amount of the D1 protein produced from each of thepsbA3andpsbA2genes. When the cells were exposed to UV‐B light, the fraction of D1 protein produced from thepsbA3gene was increased from 15–20 to 32–40% of the total D1. This effect was reversible by transferring the cells to visible light. The rate of D1 production frompsbA3increased with increasing UV‐B intensities, and was a transient phenomenon at low UV‐B levels (0.1 µE·m−2·s−1). It is concluded that the enhancement ofpsbA3gene transcription by UV‐B light leads to enhanced D1 protein synthesis from this gene. Our findings demonstrate that the main role ofpsbA3transcription activated by UV‐B is to increase the size of thepsbAmRNA pool available for translation when a rapid repair of the D1 protein is needed under UV‐B stress.
A novel function of yeast fatty acid synthase
Authors: Fichtlscherer Fabienne , Wellein Christian , Mittag Maria , Schweizer Eckhart
Published: March 2003

The prosthetic group of yeast fatty acid synthase (FAS), 4′‐phosphopantetheine, is covalently linked to Ser180 of subunit α. It originates from coenzyme A and is transferred to the enzyme by a specific phosphopantetheine:protein transferase (PPTase). The present study demonstrates that the FAS‐activating PPTase of yeast represents a distinct catalytic domain of the FAS complex and resides within the C‐terminal portion of subunit α. The autoactivation capacity of yeast FAS became evident from in vitro pantetheinylation studies using purified apo‐FAS preparations. These were readily converted to pantetheinylated holo‐FAS simply upon addition of free coenzyme A. Pantetheinylation‐competent apo‐FAS was prepared in vitro by constructing hybrid oligomers containing α‐subunits from two different pantetheine‐less FAS‐mutants. The respective mutants were selected according to their ability to complement each other, in vivo. In vitro formation of hybrid apo‐FAS complexes was achieved by dimethylmaleic anhydride (DMMA) ‐induced reversible dissociation of mixtures of the two constituent mutant enzymes. This treatment was both necessary and sufficient to produce pantetheinylation‐competent apo‐FAS. Specific FAS activities were comparable independent of whether the apo‐enzymes were pantetheinylated in vivo or in vitro. Apart from the induction of overall FAS activity, incorporation of phosphopantetheine into apo‐FAS was also demonstrated by the use of 3H‐labelled coenzyme A, leading to the formation of radioactively labelled FAS. It is concluded that pantetheinylation of yeast FAS is performed by an intrinsic catalytic activity of the apo‐enzyme proper. The endogenous PPTase acts in trans between different subunits α in the α6β6 oligomer. The self‐pantetheinylation of yeast FAS represents the first example of an apo‐enzyme being capable of post‐translational autoactivitation.
Effects of acid pH and urea on the spectral properties of the LHII antenna complex from the photosynthetic bacterium Ectothiorhodospira sp.
Authors: Buche A. , Ramirez J. M. , Picorel R.
Published: March 2003

The aim of this study was to investigate the spectral modifications of the LHII antenna complex from the purple bacterium Ectothiorhodospira sp. upon acid pH titration both in the presence and absence of urea. A blue shift specifically and reversibly affected the B850 band around pH 5.5–6.0 suggesting that a histidine residue most probably participated in the in vivo absorption red shifting mechanism. This transition was observed in the presence and absence of urea. Under strong chaotropic conditions, a second transition occurred around pH 2.0, affecting the B800 band irreversibly and the B850 reversibly. Under these conditions a blue shift from 856 to 842 nm occurred and a new and strong circular dichroism signal from the new 842 nm band was observed. Reverting to the original experimental conditions induced a red shift of the B850 band up to 856 nm but the circular dichroism signal remained mostly unaffected. Under the same experimental conditions, i.e. pH 2.1 in the presence of urea, part of the B800 band was irreversibly destroyed with concomitant appearance of a band around 770 nm due to monomeric bacteriochlorophyll from the disrupted B800. Furthermore, Gaussian deconvolution and second derivative of the reverted spectra at pH 8.0 after strong‐acid treatment indicated that the new B850 band was actually composed of two bands centered at 843 and 858 nm. We ascribed the 858 nm band to bacteriochlorophylls that underwent reversible spectral shift and the 843 nm band to oligomeric bacteriopheophytin formed from a part of the B850 bacteriochlorophyll. This new oligomer would be responsible for the observed strong and mostly conservative circular dichroism signal. The presence of bacteriopheophytin in the reverted samples was definitively demonstrated by HPLC pigment analysis. The pheophytinization process progressed as the pH decreased below 2.1, and at a certain point (i.e. pH 1.5) all bacteriochlorophylls, including those from the B800 band, became converted to oligomeric bacteriopheophytin, as shown by the presence of a single absorption band around 843 nm and by the appearance of a single main elution peak in the HPLC chromatogram which corresponded to bacteriopheophytin.