bluuebunny/crossref_metadata_2025 huggingface dataset:
Transition state complexes of the Klebsiella pneumoniae nitrogenase proteins
Authors: Miller Richard W. , Eady Robert R. , Fairhurst Shirley A. , Gormal Carol A. , Smith Barry E.
Published: March 2003

Stable inactive 2 : 1 complexes of the Klebsiella pneumoniae nitrogenase components (Kp2/Kp1) were prepared with ADP or the fluorescent ADP analogue, 2′(3′)‐O‐[N‐methylanthraniloyl] ADP and AlF4− or BeF3− ions. By analogy with published crystallographic data [Schindelin et al. (1997) Nature387, 370–376)], we suggest that the metal fluoride ions replaced phosphate at the two ATP‐binding sites of the iron protein, Kp2. The beryllium (BeFx) and aluminium (AlF4−) containing complexes are proposed to correspond to the ATP‐bound state and the hydrolytic transition states, respectively, by analogy with the equivalent complexes of myosin [Fisher et al. (1995) Biochemistry34, 8960–8972]. 31P NMR spectroscopy showed that during the initial stages of complex formation, MgADP bound to the complexed Kp2 in a manner similar to that reported for isolated Kp2. This process was followed by a second step that caused broadening of the 31P NMR signals and, in the case of the AlF4− complex, slow hydrolysis of some of the excess ADP to AMP and inorganic phosphate. The purified BeFx complex contained 3.8 ± 0.1 MgADP per mol Kp1. With the AlF4− complex, MgAMP and adenosine (from MgAMP hydrolysis) replaced part of the bound MgADP although four AlF4− ions were retained, demonstrating that full occupancy by MgADP is not required for the stability of the complex. The fluorescence emission maximum of 2′(3′)‐O‐[N‐methylanthraniloyl] ADP was blue‐shifted by 6–8 nm in both metal fluoride complexes and polarization was 6–9 times that of the free analogue. The fluorescence yield of bound 2′(3′)‐O‐[N‐methylanthraniloyl] ADP was enhanced by 40% in the AlF4− complex relative to the solvent but no increase in fluorescence was observed in the BeFx complex. Resonance energy transfer from conserved tyrosine residues located in proximity to the Kp2 nucleotide‐binding pocket was marked in the AlF4− complex but minimal in the BeFx fluoride complex, illustrating a clear conformational difference in the Fe protein of the two complexes. Our data indicate that complex formation during the nitrogenase catalytic cycle is a multistep process involving at least four conformational states of Kp2: similar to the free Fe protein; as initially complexed with detectable 31P NMR; as detected in mature complexes with no detectable 31P NMR; in the AlF4− complex in which an altered tyrosine interaction permits resonance energy transfer with 2′(3′)‐O‐[N‐methylanthraniloyl] ADP.
Mutagenesis of key residues identifies the connection subdomain of HIV‐1 reverse transcriptase as the site of inhibition by heme
Authors: Argyris Elias G. , Vanderkooi Jane M. , Paterson Yvonne
Published: March 2003

We have recently demonstrated that metalloporphyrins are potent inhibitors of both human immunodeficiency virus type 1 (HIV‐1) and human immunodeficiency virus type 2 (HIV‐2) reverse transcriptases (RTs) [Argyris, E.G., Vanderkooi, J.M., Venkateswaran, P.S., Kay, B.K., and Paterson, Y. (1999) J. Biol. Chem.274, 1549–1556]. In addition, by screening a phage peptide library we discovered that a peptide with sequence similarity to residues 398–407 from the connection subdomain of HIV RTs binds heme. These findings suggested that this highly conserved region may be the binding site for metalloporphyrins and a novel site for inhibition of enzymatic activity. Our most recent data presented here confirm this suggestion. Screening of HIV‐1 RT 398–407 peptide analogs by fluorescence assays demonstrates that Trp residues at positions 401 and 402 are important for heme binding. Furthermore, site‐directed mutagenesis of these residues verified these findings and indicated that heme inhibits HIV‐1 RT by binding on the connection subdomain of the p66 subunit of the enzyme but not on the p51 subunit. This was also confirmed by analyzing the binding affinities of heme for mutant HIV‐1 RT heterodimers, using intrinsic fluorescence assays. The clear identification of the connection domain as a novel inhibition site is crucial in understanding the mechanism of heme binding and enzymatic inhibition and will facilitate the generation of novel porphyrin‐based inhibitors of RT.
Characterization of oligosaccharides from the chondroitin sulfates
Authors: Huckerby Thomas N. , Lauder Robert M. , Brown Gavin M. , Nieduszynski Ian A. , Anderson Karen , Boocock Joanne , Sandall Patricia L. , Weeks Stephen D.
Published: March 2003

Chondroitin sulfates were fragmented using the enzymes chondroitin sulfate ABC endolyase and chondroitin ACII lyase; both disaccharide and tetrasaccharide fragments were isolated after reduction to the corresponding 2‐deoxy‐2‐N‐acetylamino‐d‐galactitol (GalNAc‐ol) form. These have the structures: ΔUA(β1–3)GalNAc4S‐ol, ΔUA(β1–3)GalNAc6S‐ol, ΔUA2S(β1–3)GalNAc6S‐ol, ΔUA(β1–3)GalNAc4S(β1–4)l‐IdoA(α1–3)GalNAc4S‐ol, ΔUA(β1–3)GalNAc4S(β1–4)GlcA(β1–3)GalNAc4S‐ol, ΔUA(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc4S‐ol, ΔUA(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S‐ol, ΔUA2S(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc4S‐ol and ΔUA2S(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S‐ol, where ΔUA represents a 4,5‐unsaturated hexuronic acid (4‐deoxy‐α‐lthreo‐hex‐4‐enepyranosyluronic acid) and 6S/4S/2S represent O‐ester sulfate groups at C6/C4/C2 sites. Complete 1H‐NMR and 13C‐NMR data are derived for these species, which may help to alleviate some of the significant difficulties resulting from signal complexity that are currently hindering the characterization and assignment of major and minor structural components within chondroitin sulfate and dermatan sulfate polymers.
Physico‐chemical requirements for cellular uptake of pAntp peptide
Authors: Drin Guillaume , Mazel Martine , Clair Philippe , Mathieu Danièle , Kaczorek Michel , Temsamani Jamal
Published: March 2003

The pAntp peptide, corresponding to the third helix of the Antennapedia homeodomain, is internalized by a receptor‐independent process into eucaryotic cells. The precise mechanism of entry remains unclear but the interaction between the phospholipids of plasma membrane and pAntp is probably involved in the translocation process. In order to define the role of peptide–lipid interaction in this mechanism and the physico‐chemical properties that are necessary for an efficient cellular uptake, we have carried out an Ala‐Scan mapping. The peptides were labeled with a fluorescent group (7‐nitrobenz‐2‐oxo‐1,3‐diazol‐4‐yl‐; NBD) and their cell association was measured by flow cytometry. Furthermore, we determined the fraction of internalized peptide by using a dithionite treatment. Comparison between cell association and cell uptake suggests that the affinity of pAntp for the plasma membrane is required for the import process. To further investigate which are the physico‐chemical requirements for phospholipid‐binding of pAntp, we have determined the surface partition coefficient of peptides by titrating them with phospholipid vesicles having different compositions. In addition, we estimated by circular dichroism the conformation adopted by these peptides in a membrane‐mimetic environment. We show that the phospholipid binding of pAntp depends on its helical amphipathicity, especially when the negative surface charge density of phospholipid vesicles is low. The cell uptake of pAntp, related to lipid‐binding affinity, requires a minimal hydrophobicity and net charge. As pAntp does not seem to translocate through an artificial phospholipid bilayer, this might indicate that it could interact with other cell surface components or enters into cells by a nonelucidated biological mechanism.
Purification, characterization and cloning of isovaleryl‐CoA dehydrogenase from higher plant mitochondria
Authors: Faivre‐Nitschke S. Emmanuelle , Couée Ivan , Vermel Matthieu , Grienenberger Jean M. , Gualberto José M.
Published: March 2003

Between the different types of Acyl‐CoA dehydrogenases (ACADs), those specific for branched chain acyl‐CoA derivatives are involved in the catabolism of amino acids. In mammals, isovaleryl‐CoA dehydrogenase (IVD), an enzyme of the leucine catabolic pathway, is a mitochondrial protein, as other acyl‐CoA dehydrogenases involved in fatty acid β‐oxidation. In plants, fatty acid β‐oxidation takes place mainly in peroxisomes, and the cellular location of the enzymes involved in the catabolism of branched‐chain amino acids had not been definitely assigned. Here, we describe that highly purified potato mitochondria have important IVD activity. The enzyme was partially purified and cDNAs from two different genes were obtained. The partially purified enzyme has enzymatic constant values with respect to isovaleryl‐CoA comparable to those of the mammalian enzyme. It is not active towards straight‐chain acyl‐CoA substrates tested, but significant activity was also found with isobutyryl‐CoA, implying an additional role of the enzyme in the catabolism of valine. The present study confirms recent reports that in plants IVD activity resides in mitochondria and opens the way to a more detailed study of amino‐acid catabolism in plant development.
Disulfide bond assignment, lipid transfer activity and secondary structure of a 7‐kDa plant lipid transfer protein, LTP2
Authors: Douliez Jean‐Paul , Pato Christine , Rabesona Hanitra , Mollé Daniel , Marion Didier
Published: March 2003

The 7‐kDa lipid transfer proteins, LTP2s, share some amino‐acid sequence similarities with the 9‐kDa isoforms, LTP1s. Both proteins display an identical cysteine motif and, in this regard, LTP2s have been classified as lipid transfer proteins. However, in contrast with LTP1s, no data are available on their structure, cysteine pairings, lipid transfer and lipid binding properties. We reported on the isolation of two isoforms of 7‐kDa lipid transfer protein, LTP2, from wheat seeds and showed for the first time that they indeed display lipid transfer activity. Trypsin and chymotrypsin digestions of the native LTP2 afforded the sequence of both isoforms and assignment of disulfide bonds. The cysteine pairings, Cys10–Cys24, Cys25–Cys60, Cys2–Cys34, Cys36–Cys67, revealed a mismatch at the Cys34‐X‐Cys36 motif of LTP2 compared to LTP1. Moreover, the secondary structure as determined by circular dichroism suggested an identical proportion of α helices, β sheets and random coils. By analogy with the structure of the LTP1, we discussed what structural changes are required to accommodate the LTP2 disulfide pattern.
Effects of mutations in mitochondrial cytochrome b in yeast and man
Authors: Fisher Nicholas , Meunier Brigitte
Published: March 2003

The mitochondrial cytochrome bc1 complex is a key protonmotive component of eukaryotic respiratory chains. The mitochondrially encoded cytochrome b forms, with cytochrome c1 and the iron–sulfur protein, the catalytic core of this multimeric enzyme. Mutations of cytochrome b have been reported in association with human diseases. In the highly homologous yeast cytochrome b, several mutations that impair the respiratory function, and reversions that correct the defect, have been described. In this paper, we re‐examine the mutations in the light of the atomic structure of the complex, and discuss the possible effect, at enzyme level, of the human cytochrome b mutations and the correcting effect of the reversions.
Tyrosine phosphorylation‐dependent activation of NF‐κB
Authors: Livolsi Antonia , Busuttil Valère , Imbert Véronique , Abraham Robert T. , Peyron Jean‐François
Published: March 2003

Phosphorylation of the N‐terminal domain of IκB inhibitory subunits induces activation of the transcription factor NF‐κB. Although serine phosphorylation has been shown to induce ubiquitination and subsequent proteasome‐mediated degradation of IκB‐α, little is known about the mechanisms that lead to release of active NF‐κB in T cells as a consequence of tyrosine phosphorylation of IκB‐α[Imbert, V., Rupec, R.A., Livolsi, A., Pahl, H.L., Traenckner, B.M., Mueller‐Dieckmann, C., Farahifar, D., Rossi, B., Auberger, P., Baeuerle, P. & Peyron, J.F. (1996) Cell86, 787–798]. The involvement of the tyrosine kinases p56lck and ZAP‐70 in this reaction is demonstrated here using specific pharmacological inhibitors and Jurkat mutants unable to express these kinases. Although the inhibitors prevented both pervanadate‐induced phosphorylation of IκB‐α on Tyr42 and NF‐κB activation, we observed that, in p56lck‐deficient Jurkat mutants, NF‐κB could still associate with IκB‐α despite phosphorylation on Tyr42. Furthermore, the SH2 domain of p56lck appeared to be required for pervanadate‐induced NF‐κB activation but not for Tyr42 phosphorylation. These results show that p56lck and ZAP‐70 are key components of the signaling pathway that leads to phosphotyrosine‐dependent NF‐κB activation in T cells and confirm that tyrosine kinases must control at least two different steps to induce activation of NF‐κB. Finally, we show that H2O2, which stimulates p56lck and ZAP‐70 in T cells, is an activator of NF‐κB through tyrosine phosphorylation of IκB‐α.
An element within the 5′ untranslated region of human Hsp70 mRNA which acts as a general enhancer of mRNA translation
Authors: Vivinus Sandra , Baulande Sylvain , van Zanten Marije , Campbell Fiona , Topley Peter , Ellis Jonathan H. , Dessen Philippe , Coste Hervé
Published: March 2003

The untranslated regions of mRNAs encoding heat‐shock proteins have been reported to contain elements important to the post‐transcriptional regulation of these key components of the stress response. In this report we describe an element from the 5′UTR of human Hsp70 mRNA that increases the efficiency of mRNA translation. Cloning of this region upstream of the coding sequence of two different reporter genes (firefly luciferase and chloramphenicol acetyltransferase) increases expression of the reporter under normal cell culture conditions by up to an order of magnitude. This effect was observed in three different promoter contexts (HSP, SV40 and CMV) and in six cell lines. The increase in protein production is not accompanied by any alteration in mRNA levels, suggesting that the element facilitates translation. 5′ or 3′ truncated sequences are ineffective in enhancing reporter expression, suggesting that the activity arises from the secondary structure of the element, rather than from some smaller defined motif. Computer analysis of this region revealed that it is able to form stable secondary structures (ΔG ≈ −292.6 kJ·mol−1). The Hsp70 element does not seem to act as an internal ribosome entry site. Incorporation of the sequence into plasmids used for DNA vaccination produces increased antibody responses, confirming that the sequence is functional in primary cells. These data suggest that the 5′UTR of human Hsp70 mRNA plays an important role in determining Hsp70 expression levels, and that it contains an element of general utility in enhancing recombinant protein expression systems.
Receptor mediated uptake of peptides that bind the human transferrin receptor
Authors: Lee Jae H. , Engler Jeffrey A. , Collawn James F. , Moore Bryan A.
Published: March 2003

A biopanning process designed to find peptide epitopes specific for cell surface receptors has been used in this study to select seven‐ and 12‐amino‐acid peptides capable of binding to and internalizing with the human transferrin receptor (hTfR). Through sequential rounds of negative and positive selection, two peptide sequences were identified that specifically bind to the hTfR. Phage containing the sequences HAIYPRH or THRPPMWSPVWP were inhibited from binding the hTfR in a dose‐dependent fashion when peptides of the same sequence were present in a competition assay. Interestingly, transferrin did not compete with either of these sequences for receptor binding, suggesting that these peptides bind a site on the hTfR distinct from the transferrin binding site. When either of these sequences was expressed as a fusion to green fluorescent protein (GFP), the recombinant GFP molecule was internalized in cells expressing the hTfR. These studies suggest that the two peptides can be used to target other proteins into the endosomal pathway. Further, they provide a strategy for identifying peptides that bind to other cell surface receptors that can be used for both diagnostic and therapeutic purposes.
A new structural type for Haemophilus influenzae lipopolysaccharide
Authors: Månsson Martin , Bauer Sebastian H. J. , Hood Derek W. , Richards James C. , Moxon E. Richard , Schweda Elke K. H.
Published: March 2003

Structural elucidation of the sialylated lipopolysaccharide (LPS) of non‐typeable Haemophilus influenzae (NTHi) strain 486 has been achieved by the application of high‐field NMR techniques and ESI‐MS along with composition and linkage analyses on O‐deacylated LPS and oligosaccharide samples. It was found that the LPS contains the common element of H. influenzae, l‐α‐d‐Hepp‐(1→2)‐[PEtn→6]‐l‐α‐d‐Hepp‐(1→3)‐[β‐d‐Glcp‐(1→4)]‐l‐α‐d‐Hepp‐(1→5)‐[PPEtn→4]‐α‐Kdop‐(2→6)‐Lipid A, but instead of glycosyl substitution of the terminal heptose residue (HepIII) at the O2 position observed in other H. influenzae strains, HepIII is chain elongated at the O3 position by either lactose or sialyllactose (i.e. α‐Neu5Ac‐(2→3)‐β‐d‐Galp‐(1→4)‐β‐d‐Glcp). The LPS is substituted by an O‐acetyl group linked to the O2 position of HepIII and phosphocholine (PCho) which was located at the O6 position of a terminal α‐d‐Glcp residue attached to the central heptose, a molecular environment different from what has been reported earlier for PCho. In addition, minor substitution by O‐linked glycine to the LPS was observed. By investigation of LPS from a lpsA mutant of NTHi strain 486, it was demonstrated that the lpsA gene product also is responsible for chain extension from HepIII in this strain. The involvement of lic1 in expression of PCho was established by investigation of a lic1 mutant of NTHi strain 486.
A paramagnetic species with unique EPR characteristics in the active site of heterodisulfide reductase from methanogenic archaea
Authors: Madadi‐Kahkesh Shahla , Duin Evert C. , Heim Steffen , Albracht Simon P. J. , Johnson Michael K. , Hedderich Reiner
Published: March 2003

Heterodisulfide reductase (Hdr) from methanogenic archaea is an iron–sulfur protein that catalyses the reversible reduction of the heterodisulfide (CoM‐S‐S‐CoB) of the methanogenic thiol coenzymes, coenzyme M (H‐S‐CoM) and coenzyme B (H‐S‐CoB). In EPR spectroscopic studies with the enzyme from Methanothermobacter marburgensis, we have identified a unique paramagnetic species that is formed upon reaction of the oxidized enzyme with H‐S‐CoM in the absence of H‐S‐CoB. This paramagnetic species can be reduced in a one‐electron step with a midpoint‐potential of −185 mV but not further oxidized. A broadening of the EPR signal in the 57Fe‐enriched enzyme indicates that it is at least partially iron based. The g values (gxyz= 2.013, 1.991 and 1.938) and the midpoint potential argue against a conventional [2Fe−2S]+, [3Fe−4S]+, [4Fe−4S]+ or [4Fe−4S]3+ cluster. This species reacts with H‐S‐CoB to form an EPR silent form. Hence, we propose that only a half reaction is catalysed in the presence of H‐S‐CoM and that a reaction intermediate is trapped. This reaction intermediate is thought to be a [4Fe−4S]3+ cluster that is coordinated by one of the cysteines of a nearby active‐site disulfide or by the sulfur of H‐S‐CoM. A paramagnetic species with similar EPR properties was also identified in Hdr from Methanosarcina barkeri.
Both the N‐ and C‐terminal chaperone sites of Hsp90 participate in protein refolding
Authors: Minami Michiko , Nakamura Miki , Emori Yasufumi , Minami Yasufumi
Published: March 2003

Hsp90 is able to bind partially unfolded firefly luciferase and maintain it in a refoldable state; the subsequent successive action of the 20S proteasome activator PA28, Hsc70 and Hsp40 enables its refolding. Hsp90 possesses two chaperone sites in the N‐ and C‐terminal domains that prevent the aggregation of denatured proteins. Here we show that both chaperone sites of Hsp90 are effective not only in capturing thermally denatured luciferase, but also in holding it in a state prerequisite for the successful refolding process mediated by PA28, Hsc70 and Hsp40. In contrast, the heat‐induced activity of Hsp90 to bind chemically denature dihydrofolate reductase efficiently and prevent its rapid spontaneous refolding was detected in the N‐terminal site of Hsp90 only, while the C‐terminal site was without effect. Thus it is most likely that both the N‐ and C‐terminal chaperone sites may contribute to Hsp90 function as holder chaperones, however, in a significantly distinct manner.
Carbohydrate binding properties of banana (Musa acuminata) lectin
Authors: Goldstein Irwin J. , Winter Harry C. , Mo Hanqing , Misaki Akira , Van Damme Els J. M. , Peumans Willy J.
Published: March 2003

This paper extends our knowledge of the rather bizarre carbohydrate binding poperties of the banana lectin (Musa acuminata). Although a glucose/mannose binding protein which recognizes α‐linked gluco‐and manno‐pyranosyl groups of polysaccharide chain ends, the banana lectin was shown to bind to internal 3‐O‐α‐d‐glucopyranosyl units. Now we report that this lectin also binds to the reducing glucosyl groups of β‐1,3‐linked glucosyl oligosaccharides (e.g. laminaribiose oligomers). Additionally, banana lectin also recognizes β1,6‐linked glucosyl end groups (gentiobiosyl groups) as occur in many fungal β1,3/1,6‐linked polysaccharides. This behavior clearly distinguishes the banana lectin from other mannose/glucose binding lectins, such as concanavalin A and the pea, lentil and Calystegia sepium lectins.
Novel analogues of neuropeptide Y with a preference for the Y1‐receptor
Authors: Söll Richard M. , Dinger Michaela C. , Lundell Ingrid , Larhammer Dan , Beck‐Sickinger Annette G.
Published: March 2003

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and acts in humans via at least three receptor subtypes: Y1, Y2, and Y5. Whereas selective agonists and antagonists are known for the Y2‐ and Y5‐receptors, the Y1‐receptor still lacks a highly selective agonist. This work presents the first NPY‐based analogues with Y1‐receptor preference and agonistic properties. Furthermore, the importance of specific amino acids of NPY for binding to the Y‐receptor subtypes is presented. Amongst the analogues tested, [Phe7,Pro34]pNPY (where pNPY is porcine neuropeptide Y) showed the most significant Y1‐receptor preference (> 1 : 3000‐fold), with subnanomolar affinity to the Y1‐receptor, and Ki values of ≈ 30 nm for the Y2‐ and Y5‐subtype, respectively. Variations of position 6, especially [Arg6,Pro34]pNPY and variations within positions 20–23 of NPY were found to result in further analogues with significant Y1‐receptor preference (1 : 400–1 : 2000). In contrast, cyclo S–S [Cys20,Cys24]pNPY was found to be a highly selective ligand at the Y2‐receptor, binding only threefold less efficiently than NPY. Analogues containing variations of positions 31 and 32 showed highly reduced affinity to the Y1‐receptor, while binding to the Y5‐receptor was affected less. Inhibition of cAMP‐accumulation of selected peptides with replacements within position 20–23 of NPY showed preserved agonistic properties. The NPY analogues tested give insights into ligand–receptor interaction of NPY at the Y1‐, Y2‐ and Y5‐receptor and contribute to our understanding of subtype selectivity. Furthermore, the Y1‐receptor‐preferring peptides are novel tools that will provide insight into the physiological role of the Y1‐receptor.
Post‐translational modifications and activation of p53 by genotoxic stresses
Authors: Appella Ettore , Anderson Carl W.
Published: March 2003

In unstressed cells, the tumor suppressor protein p53 is present in a latent state and is maintained at low levels through targeted degradation. A variety of genotoxic stresses initiate signaling pathways that transiently stabilize the p53 protein, cause it to accumulate in the nucleus, and activate it as a transcription factor. Activation leads either to growth arrest at the G1/S or G2/M transitions of the cell cycle or to apoptosis. Recent studies point to roles for multiple post‐translational modifications in mediating these events in response to genotoxic stresses through several potentially interacting but distinct pathways. The ≈ 100 amino‐acid N‐terminal and ≈ 90 amino‐acid C‐terminal domains are highly modified by post‐translational modifications. The N‐terminus is heavily phosphorylated while the C‐terminus contains phosphorylated, acetylated and sumoylated residues. Antibodies that recognize p53 only when it has been modified at specific sites have been developed, and studies with these reagents show that most known post‐translational modifications are induced when cells are exposed to genotoxic stresses. These recent results, coupled with biochemical and genetic studies, suggest that N‐terminal phosphorylations are important for stabilizing p53 and are crucial for acetylation of C‐terminal sites, which in combination lead to the full p53‐mediated response to genotoxic stresses. Modifications to the C‐terminus inhibit the ability of this domain to negatively regulate sequence‐specific DNA binding; additionally, they modulate the stability, the oligomerization state, the nuclear import/export process and the degree of ubiquitination of p53.
Regulation of p53 localization
Authors: Liang Shun‐Hsin , Clarke Michael F.
Published: March 2003

Despite intensive study of p53, the regulation of p53 cellular localization is still poorly understood. This is an overview of the elements and molecules involved in p53 nucleocytoplasmic transportation. These include the nuclear import and export signals of p53, inhibition of p53 nuclear import and export by oligomerization, MDM2‐mediated p53 nuclear export, and possible roles of p53 phosphorylation in regulating p53 cellular localization. Finally, questions regarding p53 cellular trafficking will also be discussed.
The fatty acid composition of chylomicron remnants influences their binding and internalization by isolated hepatocytes
Authors: Lambert Marc S. , Avella MichaeL. A. , Berhane Yoel , Shervill Elaine , Botham Kathleen M.
Published: March 2003

The binding and internalization of 125I‐labelled chylomicron remnants derived from palm, olive, corn, or fish oil (rich in saturated, monounsaturated, n‐6, or n‐3 polyunsaturated fatty acids, respectively) by hepatocytes from rats fed a low‐fat diet or a diet supplemented with the corresponding fat for 21 days was investigated. In hepatocytes from rats fed the low‐fat diet, the association of radioactivity with the cells at 4 °C (a measure of initial binding only) was similar with all types of remnants tested, but was more rapid at 37 °C (a measure of binding plus internalization) when fish oil, as compared to olive, corn or palm oil remnants, was used, and similar differences in the internalization of the particles were observed. In contrast, when hepatocytes from rats fed the fat‐supplemented diets were used, the rate of association at 37 °C of remnants with cells from rats fed palm, corn or fish oil was similar, and higher than that found with cells from animals fed olive oil, and in this case these differences were mainly due to changes in the binding of the particles to the cells at 4 °C. Both excess low‐density lipoprotein (LDL), which inhibits remnant uptake by the LDL receptor, and lactoferrin, which blocks the LDL receptor‐related protein (LRP), were found to decrease the association of the remnants with cells from rats fed the low‐fat and high‐fat diets. However, in hepatocytes from animals given the low‐fat diet, most of the differences between the various types of particle were retained in the presence of lactoferrin, but abolished in the presence of LDL. In contrast, in cells from rats fed the high‐fat diets, the differences were reduced by both lactoferrin and LDL. These findings demonstrate that the hepatic uptake of chylomicron remnants is influenced both by the fatty acid composition of the particles, and by longer‐term adaptive changes in liver tissue, and suggest that the former effects are mediated mainly by the LDL receptor, while the latter may involve both the LDL receptor and the LRP.
Characterization and reconstitution of functional hemagglutinin of the Clostridium botulinum type C progenitor toxin
Authors: Kouguchi Hirokazu , Watanabe Toshihiro , Sagane Yoshimasa , Ohyama Tohru
Published: March 2003

The purified progenitor toxin of Clostridium botulinum type C strain 6814 (C‐6814) forms a large complex composed of 150‐kDa neurotoxin (NT), 130‐kDa nontoxic‐nonhemagglutinin (NTNHA), and hemagglutinin (HA) components. The HA component consisted of a mixture of several subcomponents with molecular masses of 70, 55, 33, 26–21 and 17 kDa. We isolated the HA subcomponents from the progenitor toxin by chromatography in the presence of denaturants. The isolated HA subcomponents, designated as i‐HA‐33, i‐HA‐55, i‐HA‐70 and i‐HA‐33/17, were nearly homogenous on SDS/PAGE, but the HA‐17 and HA‐26–21 components were not purified. Some HA subcomponents, designated as f‐HA‐33 and f‐HA‐33/17 complex, existed free of the progenitor toxin in the culture medium and they were separately purified. Every HA subcomponent so far isolated shows binding activity to erythrocytes. The hemagglutination activities of each HA subcomponent had a titer of 25 for the f‐HA‐33/17 complex, and below 23 for the other f‐ and i‐HA subcomponents, while the parent progenitor L toxin was 28. The reconstitution of various combinations of f‐ and i‐HA subcomponents was attempted via mixing and tested for hemagglutination activity. When the i‐HA‐33/17 complex and i‐HA‐55 were mixed, the hemagglutination activity was recovered to a titer of 29, which was slightly higher than that of the parent toxin. These data imply that a combination of at least HA‐33, ‐17 and ‐55 subcomponents is required for full hemagglutination activity of the botulinum progenitor toxin, but each single HA subcomponent shows weak or no aggregation of erythrocytes.
Regulation of MDR1 promoter activity in human breast carcinoma cells by protein kinase C isozymes α and θ
Authors: Gill Parminder K. , Gescher Andreas , Gant Timothy W.
Published: March 2003

Increased levels of the protein kinase C (PKC) isoenzymes α and θ occur in conjunction with MDR1 gene expression in cells and tissues that have acquired a multidrug resistance (MDR) phenotype. Studies using PKC activators or antisense strategies against PKC suggest that activation of PKC engenders MDR1 gene transcription. In this study the potential roles of PKC‐α and PKC‐θ in MDR1 gene transcriptional regulation were explored. Human‐derived MCF‐7 breast cancer cells that lack constitutive expression of PKC‐α or PKC‐θ at detectable levels were transfected with full‐length PKC‐α or PKC‐θ genes driven by the ecdysone promoter. Stable transfectants were selected by use of the appropriate antibiotics. Treatment of these cells with ponasterone A induced expression of PKC that was catalytically active and underwent translocation and down‐regulation on exposure to 12‐O‐tetradecanoyl‐13‐phorbol acetate (TPA). These cells were used to analyse PKC‐mediated regulation of the MDR1 promoter by further transient transfection with either 1073 bp of the MDR1 gene promoter or deletion fragments thereof to −8 bp, each linked to a chloramphenicol acetyl transferase (CAT) reporter gene. In PKC‐α expressing cells TPA caused activation of all promoter fragments to −29 bp. This finding suggests that TPA‐inducible MDR1 transcription mediated through the TPA responsive factor early growth response 1 (EGR‐1) in this region of the promoter may be due to activation of PKC‐α. In contrast, PKC‐θ activated only two MDR1 fragments, −982 and −612 bp. The effect of TPA on reporter gene expression was attenuated by the PKC inhibitor GF 109203X. These data suggest that MDR1 promoter transcription can be regulated by PKC‐α and PKC‐θ. The results support the search for therapeutic strategies directed specifically against PKC‐α to ameliorate resistance of tumours against cytotoxic agents.
The effect of thiamine supplementation on tumour proliferation
Authors: Comín‐Anduix Begoña , Boren Joan , Martinez Sonia , Moro Cristina , Centelles Josep J. , Trebukhina Raisa , Petushok Nataly , Lee Wai‐Nang Paul , Boros Laszlo G. , Cascante Marta
Published: March 2003

Thiamine deficiency frequently occurs in patients with advanced cancer and therefore thiamine supplementation is used as nutritional support. Thiamine (vitamin B1) is metabolized to thiamine pyrophosphate, the cofactor of transketolase, which is involved in ribose synthesis, necessary for cell replication. Thus, it is important to determine whether the benefits of thiamine supplementation outweigh the risks of tumor proliferation. Using oxythiamine (an irreversible inhibitor of transketolase) and metabolic control analysis (MCA) methods, we measured an in vivo tumour growth control coefficient of 0.9 for the thiamine‐transketolase complex in mice with Ehrlich's ascites tumour. Thus, transketolase enzyme and thiamine clearly determine cell proliferation in the Ehrlich's ascites tumour model. This high control coefficient allows us to predict that in advanced tumours, which are commonly thiamine deficient, supplementation of thiamine could significantly increase tumour growth through transketolase activation. The effect of thiamine supplementation on tumour proliferation was demonstrated by in vivo experiments in mice with the ascites tumour. Thiamine supplementation in doses between 12.5 and 250 times the recommended dietary allowance (RDA) for mice were administered starting on day four of tumour inoculation. We observed a high stimulatory effect on tumour growth of 164% compared to controls at a thiamine dose of 25 times the RDA. This growth stimulatory effect was predicted on the basis of correction of the pre‐existing level of thiamine deficiency (42%), as assayed by the cofactor/enzyme ratio. Interestingly, at very high overdoses of thiamine, ≈ 2500 times the RDA, thiamine supplementation had the opposite effect and caused 10% inhibition of tumour growth. This effect was heightened, resulting in a 36% decrease, when thiamine supplementation was administered from the 7th day prior to tumour inoculation. Our results show that thiamine supplementation sufficient to correct existing thiamine deficiency stimulates tumour proliferation as predicted by MCA. The tumour inhibitory effect at high doses of thiamine is unexplained and merits further study.
Involvement of GFA1, which encodes glutamine–fructose‐6‐phosphate amidotransferase, in the activation of the chitin synthesis pathway in response to cell‐wall defects in Saccharomyces cerevisiae
Authors: Lagorce Arnaud , Le Berre‐Anton Veronique , Aguilar‐Uscanga Blanca , Martin‐Yken Helene , Dagkessamanskaia Adilia , François Jean
Published: March 2003

Cell‐wall damage caused by mutations of cell‐wall‐related genes triggers a compensatory mechanism which eventually results in hyperaccumulation of chitin reaching 20% of the cell‐wall dry mass. We show that activation of chitin synthesis is accompanied by a rise, from 1.3‐fold to 3.5‐fold according to the gene mutation, in the expression of most of the genes encoding enzymes of the chitin metabolic pathways. Evidence that GFA1, which encodes glutamine–fructose‐6‐Phosphate amidotransferase (Gfa1p), the first committed enzyme of this pathway, plays a major role in this process was as follows. Activation of chitin synthesis in the cell‐wall mutants correlated with activation of GFA1 and with a proportional increase in Gfa1p activity. Overexpression of GFA1 caused an approximately threefold increase in chitin in the transformed cells, whereas chitin content was barely affected by the joint overexpression of CHS3 and CHS7. Introduction of a gfa1‐97 allele mutation in the cell‐wall‐defective gas1Δ mutant or cultivation of this mutant in a hyperosmotic medium resulted in reduction in chitin synthesis that was proportional to the decrease in Gfa1p activity. Finally, the stimulation of chitin production was also accompanied by an increase in pools of fructose 6‐Phosphate, a substrate of Gfa1p. In quantitative terms, we estimated the flux‐coefficient control of Gfa1p to be in the range of 0.90, and found that regulation of the chitin metabolic pathway was mainly hierarchical, i.e. dominated by regulation of the amount of newly synthesized GFA1 protein. In the search for the mechanism by which GFA1 is activated in response to cell‐wall perturbations, we could only show that neither MCM1 nor RLM1, which encode two transcriptional factors of the MADS box family that are required for expression of cell‐cycle and cell‐wall‐related genes, was involved in this process.
Investigation of a functional requirement for isoprenylation by the human prostacyclin receptor
Authors: Miggin Sinead M. , Lawler Orlaith A. , Kinsella B. Therese
Published: March 2003

In the current study, we have established that the human (h) prostacyclin receptor (IP) is isoprenylated in whole cells. Through site directed mutagenesis and generation of the isoprenylation defective hIPSSLC, it was established that while isoprenylation of hIP does not influence ligand binding, it is obligatory for agonist activation of adenylyl cyclase and cAMP generation. Overexpression of GαS significantly augmented cAMP generation by the hIP but not by the hIPSSLC. Moreover, GαS co‐immunoprecipitated with hIP following agonist activation but did not co‐immunoprecipitate with hIPSSLC. Whereas hIP mediated concentration‐dependent activation of phospholipase C (PLC); the extent of PLC activation by hIPSSLC was impaired compared to hIP. Co‐expression of Gαq significantly augmentated intracellular calcium mobilization by the hIP but not by hIPSSLC. Moreover, whereas Gαq co‐immunoprecipitated with hIP, it failed to co‐immunoprecipitate with hIPSSLC. While both the hIP and hIPSSLC underwent agonist‐induced internalization, the kinetics and extent of hIPSSLC internalization was impaired compared to hIP. Altering the CAAX motif of the hIP from a farnesyl (–CSLC) to a geranylgeranyl (–CSLL) isoprene acceptor, to generate hIPCSLL, did not affect ligand binding and yielded a receptor that exhibited identical signalling through both Gs‐ and Gq‐coupled effectors to that of hIP.

Thus, whereas isoprenylation of hIP does not influence ligand binding, it is functionally imperative in regulating post‐receptor events including agonist‐activation of adenylyl cyclase, for efficient activation of PLC and for receptor internalization. Though the nature of the isoprenoid attached to hIP does not act as a major determinant, the presence of an isoprenoid group, for example farnesyl or geranylgeranyl, is required for functional receptor–G protein interaction and coupling and for efficient agonist‐ induced receptor internalization.
Lipopolysaccharide regions involved in the activation of Escherichia coli outer membrane protease OmpT
Authors: Kramer R. Arjen , Brandenburg Klaus , Vandeputte‐Rutten Lucy , Werkhoven Marjolein , Gros Piet , Dekker Niek , Egmond Maarten R.
Published: March 2003

OmpT is an integral outer membrane protease of Escherichia coli. Overexpression of OmpT in E. coli and subsequent in vitro folding of the produced inclusion bodies yielded protein with a native‐like structure. However, enzymatically active protease was only obtained after addition of the outer membrane lipid lipopolysaccharide (LPS). OmpT is the first example of an enzyme that requires LPS for activity. In this study, we investigated the nature of this activation. Circular dichroism analysis showed that binding of LPS did not lead to large structural changes. Titration of OmpT with LPS and determining the resulting OmpT activity with a fluorimetric assay yielded a dissociation constant of 10−4 m for E. coli K‐12 LPS. Determining the dissociation constants for different LPS chemotypes revealed that a fully acylated lipid A part is minimally required for activation of OmpT. The heptose‐bound phosphates in the inner core region were also important for activation. The affinity for LPS was not dependent on the concentration of substrate, neither was affinity for the substrate influenced by the concentration of LPS. This indicated that LPS most likely does not act at the level of substrate binding. We hypothesize that LPS induces a subtle conformational change in the protein that is required for obtaining a native active site geometry.
Differential effects of ergosterol and cholesterol on Cdk1 activation and SRE‐driven transcription
Authors: Suárez Yajaira , Fernández Carlos , Ledo Beatriz , Ferruelo Antonio J. , Martín Miguel , Vega Miguel A. , Gómez‐Coronado Diego , Lasunción Miguel A.
Published: March 2003

Cholesterol is essential for cell growth and division, but whether this is just a consequence of its use in membrane formation or whether it also elicits regulatory actions in cell cycle machinery remains to be established. Here, we report on the specificity of this action of cholesterol in human cells by comparing its effects with those of ergosterol, a yeast sterol structurally similar to cholesterol. Inhibition of cholesterol synthesis by means of SKF 104976 in cells incubated in a cholesterol‐free medium resulted in cell proliferation inhibition and cell cycle arrest at G2/M phase. These effects were abrogated by cholesterol added to the medium but not by ergosterol, despite that the latter was used by human cells and exerted similar homeostatic actions, as the regulation of the transcription of an SRE‐driven gene construct. In contrast to cholesterol, ergosterol was unable to induce cyclin B1 expression, to activate Cdk1 and to resume cell cycle in cells previously arrested at G2. This lack of effect was not due to cytotoxicity, as cells exposed to ergosterol remained viable and, upon supplementing with UCN‐01, an activator of Cdk1, they progressed through mitosis. However, in the presence of suboptimal concentrations of cholesterol, ergosterol exerted synergistic effects on cell proliferation. This is interpreted on the basis of the differential action of these sterols, ergosterol contributing to cell membrane formation and cholesterol being required for Cdk1 activation. In summary, the action of cholesterol on G2 traversal is highly specific and exerted through a mechanism different to that used for cholesterol homeostasis, reinforcing the concept that cholesterol is a specific regulator of cell cycle progression in human cells.
The Function of Male Dominance in the Eusocial Wasp, Mischocyttarus mastigophorus (Hymenoptera: Vespidae)
Authors: O’Donnell Sean
Published: March 2003

Males of a Neotropical eusocial wasp, Mischocyttarus mastigophorus, are dominant over their female nest mates. Mischocyttarus mastigophorus males behave aggressively toward females, while females rarely bite or chase males. Aggressive interactions between the sexes are behaviorally indistinguishable from dominance interactions among females. Males are long‐lived as adults, and can reside on nests for periods of at least one month. Furthermore, males comprise a large proportion of post‐emergence colony populations throughout much of the colony cycle. Males on the nest perform maintenance tasks at low rates, but contribute little other labor to their colonies. Males do not forage, but consume a disproportionate amount of the food (nectar and insect prey) collected by workers. Males in some colonies direct disproportionate amounts of aggression toward their queens, which may further contribute to males’ food procurement. These data suggest that adult males represent a considerable energetic and labor cost to their colonies. I hypothesize that the dominance structure of M. mastigophorus directs food resources to adult males, with the function of increasing their longevity. Increased male longevity may be selectively advantageous in tropical species such as M. mastigophorus that found new colonies throughout much or all of the year. When females initiate new nests over much of the year, individual males’ mating opportunities may be temporally distributed, favoring longer adult lifespans. Male dominance is predicted to occur in other populations of independent‐founding Neotropical Polistinae with asynchronous colony foundation.
Threat‐sensitive Predator Avoidance by Larval Pacific Treefrogs (Amphibia, Hylidae)
Authors: Puttlitz Matthew H. , Chivers Douglas P. , Kiesecker Joseph M. , Blaustein Andrew R.
Published: March 2003

According to the threat‐sensitive predator avoidance hypothesis, the intensity of a prey animal’s antipredator response should reflect its vulnerability to a specific predator. In laboratory experiments, we observed the intensity of antipredator responses of Pacific treefrog (Hyla regilla) tadpoles to stimuli from caged larval northwestern salamander (Ambystoma gracile) predators. We varied the sizes of the tadpoles relative to the salamanders in an attempt to create differences in vulnerability of tadpoles to the salamander predators. After documenting the response of the tadpoles to the caged predator, we tested the tadpole’s vulnerability to the predator by releasing the tadpole with the predator. We observed that as the relative size of the tadpoles to the caged salamanders increased, the antipredator response of the tadpoles decreased. These changes in behaviour closely mirrored changes in actual vulnerability to the predator. Our results provide experimental support for the threat‐sensitive predator avoidance hypothesis.
Lekking in Gryllotalpa major, the Prairie Mole Cricket (Insecta: Gryllotalpidae)
Authors: Hill Peggy S. M.
Published: March 2003

Gryllotalpa major is a rare, burrowing insect native to the tallgrass prairie of the south‐central United States and is known to exhibit ‘lek‐like’ behavior during mating. Here I report on a study carried out in the field that demonstrates that the prairie mole cricket meets all criteria defining a classical lekking species. Males construct specialized acoustic burrows from which they call to attract females for mating. I show that these burrows, which seem to serve no purpose other than for sexual advertisement and mating, are aggregated spatially on at least three scalar levels. Females fly through the aggregation of burrows and drop to the ground in the vicinity of calling males, and are, thus, not constrained in choosing a mate. Females enter the males’ acoustic burrows, but I argue that the burrows are not used as oviposition sites, and that the males do not otherwise sequester resources important to females. Although the term ‘lek’ is useful for the discussion of mating systems, its definition remains ambiguous. I discuss the current usage of the term and suggest extensions.
Co‐operative Rearing by Slender‐tailed Meerkats (Suricata suricatta) in the Southern Kalahari
Authors: Doolan S. P. , Macdonald D. W.
Published: March 2003

Slender‐tailed meerkats or suricates, Suricata suricatta, are small, gregarious and largely insectivorous mongooses which inhabit the arid and semi‐arid areas of southern Africa. We describe four elements of co‐operative rearing of the young: 1. babysitting at the den; 2. creching on foraging trips; 3. provisioning with prey items away from the den; and 4. allonursing, including spontaneous lactation. Individual band members differed markedly in contributions to co‐operative rearing. All adults and yearlings guarded the den and helpers actively defended young against predators. Breeders babysat significantly less than did non‐breeding adults. Differences in age–sex class contributions were also evident in creching behaviour. The feeding of kittens by provisioners extended from soon after their first emergence from the den at 2–3 wk of age to effective foraging independence at 10–12 wk. Breeding females initially contributed little care other than nursing. Allonursing occurred in six of 25 closely observed litters, including three incidents of spontaneous lactation when subordinates nursed the young of higher‐ranking females. Such co‐operation is likely to be critical to the survival of these highly sociable mongooses in a semi‐arid environment such as the Kalahari where food availability can fluctuate enormously.