bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective labelling of diazepam‐insensitive GABAA receptors in vivo using [3H]Ro 15‐4513
Authors: Pym Luanda J , Cook Susan M , Rosahl Thomas , McKernan Ruth M , Atack John R
Published: September 2005

Classical benzodiazepines (BZs), such as diazepam, bind to GABAA receptors containing α1, α2, α3 or α5 subunits that are therefore described as diazepam‐sensitive (DS) receptors. However, the corresponding binding site of GABAA receptors containing either an α4 or α6 subunit do not bind the classical BZs and are therefore diazepam‐insensitive (DIS) receptors; a difference attributable to a single amino acid (histidine in α1, α2, α3 and α5 subunits and arginine in α4 and α6).

Unlike classical BZs, the imidazobenzodiazepines Ro 15‐4513 and bretazenil bind to both DS and DIS populations of GABAA receptors. In the present study, an in vivo assay was developed using lorazepam to fully occupy DS receptors such that [3H]Ro 15‐4513 was then only able to bind to DIS receptors.

When dosed i.v., [3H]Ro 15‐4513 rapidly entered and was cleared from the brain, with approximately 70% of brain radioactivity being membrane‐bound. Essentially all membrane binding to DS+DIS receptors could be displaced by unlabelled Ro 15‐4513 or bretazenil, with respective ID50 values of 0.35 and 1.2 mg kg−1.

A dose of 30 mg kg−1 lorazepam was used to block all DS receptors in a [3H]Ro 15‐1788 in vivo binding assay. When predosed in a [3H]Ro 15‐4513 binding assay, lorazepam blocked [3H]Ro 15‐4513 binding to DS receptors, with the remaining binding to DIS receptors accounting for 5 and 23% of the total (DS plus DIS) receptors in the forebrain and cerebellum, respectively.

The in vivo binding of [3H]Ro 15‐4513 to DIS receptors in the presence of lorazepam was confirmed using α1H101R knock‐in mice, in which α1‐containing GABAA receptors are rendered diazepam insensitive by mutation of the histidine that confers diazepam sensitivity to arginine. In these mice, and in the presence of lorazepam, there was an increase of in vivo [3H]Ro 15‐4513 binding in the forebrain and cerebellum from 4 and 15% to 36 and 59% of the total (i.e. DS plus DIS) [3H]Ro 15‐4513 binding observed in the absence of lorazepam.

British Journal of Pharmacology (2005) 146, 817–825. doi:10.1038/sj.bjp.0706392
Subtype‐specific actions of β‐amyloid peptides on recombinant human neuronal nicotinic acetylcholine receptors (α7, α4β2, α3β4) expressed in Xenopus laevis oocytes
Authors: Pym Luanda , Kemp Mark , Raymond‐Delpech Valérie , Buckingham Steven , Boyd C A R , Sattelle David
Published: September 2005

Two‐electrode voltage‐clamp electrophysiology has been used to study the actions of two amyloid peptides (Aβ1–42, Aβ1–40) on α7, α4β2 and α3β4 recombinant human neuronal nicotinic acetylcholine receptors (nicotinic AChRs), heterologously expressed in Xenopus laevis oocytes.

The application of Aβ1–42 or Aβ1–40 (1 pM–100 nM) for 5 s does not directly activate expressed human α7, α4β2 or α3β4 nicotinic AChRs.

Aβ1–42 and Aβ1–40 are antagonists of α7 nicotinic AChRs. For example, 10 nM Aβ1–42 and Aβ1–40 both reduced the peak amplitude of currents recorded (3 mM ACh) to 48±5 and 45±10% (respectively) of control currents recorded in the absence of peptide. In both the cases the effect is sustained throughout a 30 min peptide application and is poorly reversible.

Aβ1–42 and Aβ1–40 (10 nM) enhance currents recorded in response to ACh (3 mM) from oocytes expressing α4β2 nicotinic AChRs by 195±40 and 195±41% respectively. This effect is transient, reaching a peak after 3 min and returning to control values after a 24 min application of 10 nM Aβ1–42. We observe an enhancement of 157±22% of control ACh‐evoked current amplitude in response to 100 nM Aβ1–42 recorded from oocytes expressing α4β2 nicotinic AChRs.

Aβ1–42 and Aβ1–40 (10 nM) were without antagonist actions on the responses of α3β4 nicotinic AChRs to ACh (1 nM–3 mM).

British Journal of Pharmacology (2005) 146, 964–971. doi:10.1038/sj.bjp.0706403
Structural aspects of histone H1–DNA complexes and their relation to transfection efficiency
Authors: Haberland Annekathrin , Cartier Régis , Heuer Diane , Zaitsev Sergei , Paulke Bernd‐R. , Schäfer‐Korting Monika , Böttger Michael
Published: September 2005

During transfection, polycation–DNA complexes are normally diluted by the transfection medium, which often contains salt in the physiological concentration range and serum. It is not exactly known to what extent this dilution step influences the properties of the complexes, which in turn influence the transfection efficiency. In order to gain more insight into the size–structure–transfection activity relationship, we prepared histone H1–DNA complexes in NaCl solutions at various concentrations known to determine the size and structure of the resulting complexes. We characterized the complexes by physicochemical methods. Fluorescence correlation spectroscopy enabled relative measurements of complex sizes even under physiological conditions. The different appearances of the complexes were correlated with their transfection efficiency. When transfection was performed by dilution of the complexes in cell‐cultivation media, the initial structure of H1–DNA complexes preformed under distinct salt conditions had no significant influence on the transfection efficiency. The dilution of the preformed complexes with cell‐cultivation medium resulted in re‐formation and aggregation of the complexes. The addition of the complexes to the cells without cell‐cultivation medium, however, showed a direct correlation between the size of the complexes and the transfection efficiency (corrrelation coefficient 0.91). Small complexes did not contribute to the transfection.
Double mutant of Aspergillus oryzae for improved production of l‐dopa (3,4‐dihydroxyphenyl‐l‐alanine) from l‐tyrosine
Authors: Ali Sikander , Ikram‐ul‐Haq  , Qadeer Muhammad A. , Rajoka Muhammad I.
Published: September 2005

Aspergillus oryzae mutant strain UV‐7 was further improved for the production of l‐dopa from l‐tyrosine using chemical mutation. Different putative mutant strains of the organism were tested for the production of l‐dopa in triplicate shake‐flask cultures. Among these putative mutants, the strain designated SI‐12 gave a maximal production of l‐dopa (444±14 mg of l‐dopa/g of cells). The regulation of l‐dopa from different carbon source solutions [initial substrate concentration (S0)=30 g/l] by the mutant culture was investigated. At an initial pH (pH0) of 5.0 and a temperature (T) of 30 °C, 100% of sugars were utilized for product and cell mass formation, corresponding to final l‐dopa product yield of 189±8 mg/g of substrate utilized and maximum volumetric and specific productivities of 145±5 mg/h per litre and 155±8 mg/h per g of cells respectively. There was up to 3‐fold enhancement in product formation rate. This enhancement is, to our knowledge, the highest reported in the literature. To explain the kinetic mechanism of l‐dopa formation and its thermal inactivation, the thermodynamic parameters were determined with the application of the Arrhenius model. Activation enthalpy and entropy for product formation, in the case of the mutant derivative, were 40 kJ/mol and 0.076 kJ·mol−1·K−1 for its production and 116 kJ/mol and 0.590 kJ·mol−1·K−1 for thermal inactivation respectively. The respective values for product formation and product de‐activation were lower than the respective values for the parental culture. Therefore the mutant strain was thermodynamically more resistant to thermal denaturation during the product‐formation process.
A factorial experiment for optimizing the PCR conditions in routine genotyping
Authors: Niens Marijke , Spijker Geert T. , Diepstra Arjan , Te Meerman Gerard J.
Published: September 2005

Although most PCRs would produce proper PCR products when first tried, a general optimization is required to yield the best results. This optimization is often achieved by changing one factor at a time. However, this may lead to suboptimal results, since interactions between conditions are difficult to detect with this approach. In the present study, we describe the factorial optimization of PCR conditions for microsatellite genotyping, by introducing small systematic variations in conditions during the genotyping process. The hypothesis was that small changes will not affect genotyping results, but will provide information about the optimality of current conditions. The conditions to vary were the concentrations of buffer, MgCl2, dNTPs, primers, Taq polymerase and DNA, the annealing temperature (Ta) and the number of cycles. We show that, by a 28 factorial experiment, it is possible to identify not only the factors responsible for obtaining good results, but also those responsible for bad results. However, the condition leading to the highest signals is not necessarily the best operational condition. The best operational condition is minimally sensitive to random pipetting fluctuations and yields reliable genotypes as well.
Clonal evolution of stem and differentiated cells can be predicted by integrating cell‐intrinsic and ‐extrinsic parameters
Authors: Viswanathan Sowmya , Davey Ryan E. , Cheng Donavan , Raghu Riyad C. , Lauffenburger Douglas A. , Zandstra Peter W.
Published: September 2005

Stem cells and their derivatives represent a renewable source of cells for therapeutic applications. However, the inability to quantitatively integrate and exploit the effects of multiple parameters on the fate of stem cells limits their use in clinical applications. To address this, we developed a computational model that combines probabilistic, individual‐cell and deterministic cell‐population parameters to simultaneously calculate the specific effects of exogenous and endogenous factors on the overall population‐dynamics behaviour. The model tracks the progeny trajectory of individual cells over several generations as a threshold function of ligand–receptor signalling interactions. Simulations in silico were validated against an Oct4‐promoter‐driven green‐fluorescent‐protein‐expressing murine embryonic stem cell line, and used to understand the effects of key parameters on the clonal evolution of stem versus differentiated cells in this system. Our approach demonstrated the ability to distinguish between individual‐cell and population‐averaged parameters with respect to their effects on governing dynamic behaviour. Moreover, we could discriminate between digital versus graded regulation of the Oct4 transcription factor in accounting for experimental observations. Finally, we showed that our approach could be generalized to other stem‐cell systems, in particular the previously characterized intestinal crypt system, in elucidating relative contributions of stem and progenitor cells to population output. On the basis of all these results, we believe that our iterative experimental and computational approach has been found to be useful for the study of various stem‐cell systems.
Transdermal delivery of interferon‐γ (IFN‐γ) mediated by penetratin, a cell‐permeable peptide
Authors: Lee Jongsung , Jung Eunsun , Park Junho , Park Deokhoon
Published: September 2005

IFN‐γ (interferon‐γ) has several applications in the treatment of IFN‐γ‐related skin disorders. While systemic delivery – the major route used to administer IFN‐γ – results in significant side effects and toxicity, including fever, fatigue, nausea, vomiting and neurotoxicity, transdermal delivery has a very low transduction efficiency. In order to improve the efficiency of transdermal IFN‐γ delivery, we introduced a Pen (penetratin) peptide, a 16‐amino‐acid‐long polypeptide corresponding to the third helix of the DNA‐binding domain (homoeodomain) of Antennapedia (a Drosophila transcription factor). The human IFN‐γ gene was then fused with a gene fragment that encodes the Pen of Antennapedia in a bacterial expression vector, producing a genetic in‐frame Pen–IFN‐γ. The expressed and purified Pen–IFN‐γ was then found to have a much more efficient transduction profile than native IFN‐γ. In addition, compared with native IFN‐γ, Pen–IFN‐γ exhibited similar activities when added exogenously to a culture medium: (i) induction of IRF‐1 gene expression, and (ii) NF‐κB (nuclear factor κB) luciferase reporter activation. These results indicate that the transdermal delivery system using Pen may be an excellent way to replenish IFN‐γ in the various disorders related to this cytokine.
Development of a novel serum‐free freezing medium for mammalian cells using the silk protein sericin
Authors: Sasaki Masahiro , Kato Yoichi , Yamada Hideyuki , Terada Satoshi
Published: September 2005

Cryopreservation is a pivotal process in cellular engineering for creating a continuous source of generated functional cell lines and for the convenience of various medical treatments that involve cell culture. FBS (fetal bovine serum) supplemented with 10% (v/v) DMSO is extensively used as a freezing medium for mammalian cells using conventional methods. However, FBS should ideally be avoided, owing to serious concerns regarding bovine spongiform encephalopathy and other infections such as viruses, and an alternative to FBS is eagerly awaited. Furthermore, biomedicines and cells for transplantation should not be infectious. The present study aimed to develop a novel serum‐free freezing medium. For this purpose, we focused on using the silk protein sericin as a cryoprotectant for storage and developed a novel serum‐free freezing medium consisting of PBS, 1% (v/w) sericin, 0.5% (v/w) maltose, 0.3% (v/w) proline, 0.3% (v/w) glutamine and 10% DMSO. This novel freezing medium was compared with the conventional FBS supplemented with DMSO and also with three purchased freezing media with respect to cryopreservation of the P3U1 myeloma cell line and Chinese‐hamster ovary cells. As a result, the constructed medium containing sericin successfully cryopreserved both cell types as efficiently as the conventional medium of FBS containing 10% DMSO and was superior to all three of the purchased media. The constructed medium containing sericin also cryopreserved normal human dermal fibroblasts, human epidermal keratinocytes, the rat phaeochromocytoma cell line PC12 and insect (Spodoptera frugiperda) cell line Sf9 as effectively as the conventional medium of FBS and DMSO.
Identification of trypanothione from the human pathogen Entamoeba histolytica by mass spectrometry and chemical analysis
Authors: Ondarza Raúl N. , Hurtado Gerardo , Iturbe Angélica , Hernández Eva , Tamayo Elsa , Woolery Mathew
Published: September 2005

In this paper, we present definitive data to show, from ESI (electrospray ionization) studies, that the thiol–bimane compound isolated and purified from Entamoeba histolytica trophozoites, corresponds unequivocally to the structure of trypanothione. Trypanothione disulphide was shown to have a molecular ion of m/z 722. It was further demonstrated by MALDI–TOF (matrix‐assisted laser desorption ionization–time‐of‐flight) MS that this thiol compound also corresponds to the characteristic monoprotonated ion of trypanothione‐(bimane)2, which has a molecular ion of m/z 1103.95. The ion pattern of the thiol–bimane compound prepared from the commercial trypanothione standard is identical with the E. histolytica thiol–bimane compound. After HPLC separation, chemical amino acid analysis by dabsylation and dansylation of the thiol bimane compound from Entamoeba showed the presence of the following trypanothione components: glutamic acid, cysteic acid, glycine and spermidine. We can conclude from these highly reliable MS experiments and chemical analyses that E. histolytica contains the thiol compound trypanothione, which was previously thought to occur only in trypanosomatids.
Normal‐flow virus filtration: detection and assessment of the endpoint in bioprocessing
Authors: Bolton Glen , Cabatingan Mark , Rubino Mike , Lute Scott , Brorson Kurt , Bailey Mark
Published: September 2005

The breakthrough of a model virus, bacteriophage ΦX‐174, through normal‐flow virus filters was studied using both commercial process fluids and model feedstreams. The results indicate that (i) ΦX‐174 is a reasonable model for a mammalian parvovirus [MMV (murine minute virus)] in virus filtration studies; (ii) ΦX‐174 LRV [log(reduction value)] shows a better correlation with percentage flow decline compared with volume processed under a variety of conditions; (iii) although the extent of decline in virus LRV is dependent on the mechanism of filter fouling, the fouling mechanisms operative in a viral validation study are representative of those likely to be found under actual production conditions. The mechanism of LRV decline by many process streams was proposed to be due to selective plugging of small pores. A theoretical model as well as a predictive equation for LRV decline versus flow decay was derived; experimental results from filtration studies using pore‐plugging feedstocks were consistent with the equation. As protein solutions may vary in their adsorptive versus plugging behaviour during filtration, an evaluation of the LRV‐versus‐flow‐decay relationship on a biopharmaceutical‐product‐specific basis may be warranted.
Argatroban‐coupled Affi‐Gel matrix for the purification of thrombin from plasma
Authors: Lefkowitz Jerry B.
Published: September 2005

Sometimes it is necessary to obtain thrombin from limited amounts of human plasma for laboratory assay. None of the available purification methods easily deals with this subject. The procedure described in the present paper uses a readily available pharmaceutical agent, argatroban, to construct an affinity matrix. Argatroban has a high affinity for thrombin and its thrombin binding is reversible. Prothrombin derived from a Ba2+ precipitate of human plasma is used as the starting material. The crude prothrombin can be bulk activated to thrombin using taipan‐snake (Oxyuranus scutellatus) venom and bound to the argatroban‐coupled matrix without further processing steps. The thrombin product eluted from the argatroban matrix is very pure as judged by high specific activity and by electrophoresis. This purification scheme is rapid, yielding purified thrombin within 2 days.
Nuclear proteins as gene‐transfer vectors
Authors: Haberland Annekathrin , Böttger Michael
Published: September 2005

An interest in nuclear proteins as possible gene vectors arose as early as 1988. Nuclear proteins possess important features, such as DNA condensing capacity and the presence of NLSs (nuclear localization signals), which are thought to be perfect tools to combine cellular and nuclear uptake of DNA. The NLS of the proteins might support the intracellular trafficking of the complexes, whereas their condensing capacity enables DNase stability. Nuclear proteins, histones and also non‐histones, such as the high‐mobility‐group proteins, have been successfully employed in in vitro gene transfer by a variety of workers over the last decade. In the pertinent published data, almost all nuclear proteins tested showed gene‐transfer activity. The degree of activity of the individual nuclear proteins tested varied according to sequential and structural differences of the proteins, discriminating their DNA binding behaviour, but reported transfection efficiency differences are inconsistent. They do not reveal a logical pattern, such as a correlation with their DNA affinity. The differences in transfection efficiency of certain nuclear proteins reported by different authors could be based on a lot of factors, including the source or method of protein preparation, the cells used, the media or the transfection additives, such as lysosomotropic agents. The variances are quite random, which suggests an involvement of physicochemical parameters of the nuclear protein–DNA complexes in the efficiency of gene expression. Physicochemical features of the complexes are also determined by factors such as transfection additives (e.g. Ca2+). The present review therefore includes, in addition to an overview about the nuclear proteins used in gene‐transfer experiments, some assumptions about the mechanism of the cellular uptake of nuclear protein–DNA complexes and their translocation to the nucleus.
Regulation of plant aquaporin activity
Authors: Chaumont François , Moshelion Menachem , Daniels Mark J.
Published: September 2005

Accumulating evidence indicates that aquaporins play a key role in plant water relations. Plant aquaporins are part of a large and highly divergent protein family that can be divided into four subfamilies according to amino acid sequence similarity. As in other organisms, plant aquaporins facilitate the transcellular movement of water, but, in some cases, also the flux of small neutral solutes across a cellular membrane. Plant cell membranes are characterized by a large range of osmotic water permeabilities, and recent data indicate that plant aquaporin activity might be regulated by gating mechanisms. The factors affecting the gating behaviour possibly involve phosphorylation, heteromerization, pH, Ca2+, pressure, solute gradients and temperature. Regulation of aquaporin trafficking may also represent a way to modulate membrane water permeability. The aim of this review is to integrate recent molecular and biophysical data on the mechanisms regulating aquaporin activity in plant membranes and to relate them to putative changes in protein structure.
DSP1, a Drosophila HMG protein, is involved in spatiotemporal expression of the homoeotic gene Sex combs reduced
Authors: Rappailles Aurélien , Decoville Martine , Locker Daniel
Published: September 2005

Background information. The Pc‐G (Polycomb group) and trx‐G (trithorax group) genes play a key role in the regulation of the homoeotic genes. The homoeotic gene Scr (Sex combs reduced) contained in the Antennapedia complex specifies segmental identity of the labial and prothoracic segments in Drosophila. Regulation of Scr requires the action of different enhancer elements spread over several kilobases. We previously identified an HMGB (high mobility group)‐like protein DSP1 (dorsal switch protein 1), which works like a trx‐G protein for the normal Scr expression.

Results. In the present study, we attempted to characterize the regulatory sequences involved in the maintenance of the Scr activation by DSP1. We report here, using a transgenic line for the Scr10.0XbaI‐regulatory element, that lack of DSP1 affects the function of a reporter gene in legs' imaginal discs but not in embryos. We show by immunolocalization that DSP1 is recruited on polytene chromosomes to the insertion site of the transgene. Moreover, using chromatin immunoprecipitation experiments, we identify two regions of 1 kb in Scr10.0XbaI as the main DSP1 targets.

Conclusion. These results provide strong evidence that the Scr gene expression is influenced by direct interaction between DSP1 and two Scr regulation elements. In addition, our results show that this interaction undergoes dynamic changes during development.
Participation of both Gab1 and Gab2 in the activation of the ERK/MAPK pathway by epidermal growth factor
Authors: Meng Songshu , Chen Zhengming , Munoz-Antonia Teresita , Wu Jie
Published: September 2005

Three members of Gab family docking proteins, Gab1, Gab2 and Gab3, have been identified in humans. Previous studies have found that the hepatocyte growth factor preferentially utilizes Gab1 for signalling, whereas Bcr-Abl selectively signals through Gab2. Gab1–SHP2 interaction has been shown to mediate ERK (extracellular-signal-regulated kinase) activation by EGF (epidermal growth factor). However, it was unclear whether EGF selectively utilizes Gab1 for signalling to ERK and whether Gab2 is dispensable in cells where Gab1 and Gab2 are co-expressed. Using T47D and MCF-7 human breast carcinoma cells that express endogenous Gab1 and Gab2, we examined the role of these docking proteins in EGF-induced ERK activation. It was found that EGF induced a similar amount of SHP2–Gab1 and SHP2–Gab2 complexes. Expression of either SHP2-binding defective Gab1 or Gab2 mutant blocked EGF-induced ERK activation. Down-regulation of either Gab1 or Gab2 by siRNAs (small interfering RNAs) effectively inhibited the EGF-stimulated ERK activation pathway and cell migration. Interestingly, the inhibitory effect of Gab1 siRNA could be rescued not only by expression of an exogenous mouse Gab1 but also by an exogenous human Gab2 and vice versa, but not by IRS1 (insulin receptor substrate 1). These results reveal that Gab2 plays a pivotal role in the EGF-induced ERK activation pathway and that it can complement the function of Gab1 in the EGF signalling pathway. Furthermore, Gab1 and Gab2 are critical signalling threshold proteins for ERK activation by EGF.
Regulation of cell survival by lipid phosphate phosphatases involves the modulation of intracellular phosphatidic acid and sphingosine 1-phosphate pools
Authors: Long Jaclyn , Darroch Peter , Wan Kah Fei , Kong Kok Choi , Ktistakis Nicholas , Pyne Nigel J. , Pyne Susan
Published: September 2005

We have shown previously that LPPs (lipid phosphate phosphatases) reduce the stimulation of the p42/p44 MAPK (p42/p44 mitogen-activated protein kinase) pathway by the GPCR (G-protein-coupled receptor) agonists S1P (sphingosine 1-phosphate) and LPA (lysophosphatidic acid) in serum-deprived HEK-293 cells [Alderton, Darroch, Sambi, McKie, Ahmed, N. J. Pyne and S. Pyne (2001) J. Biol. Chem. 276, 13452–13460]. In the present study, we now show that this can be blocked by pretreating HEK-293 cells with the caspase 3/7 inhibitor, Ac-DEVD-CHO [N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde)]. Therefore LPP2 and LPP3 appear to regulate the apoptotic status of serum-deprived HEK-293 cells. This was supported further by: (i) caspase 3/7-catalysed cleavage of PARP [poly(ADP-ribose) polymerase] was increased in serum-deprived LPP2-overexpressing compared with vector-transfected HEK-293 cells; and (ii) serum-deprived LPP2- and LPP3-overexpressing cells exhibited limited intranucleosomal DNA laddering, which was absent in vector-transfected cells. Moreover, LPP2 reduced basal intracellular phosphatidic acid levels, whereas LPP3 decreased intracellular S1P in serum-deprived HEK-293 cells. LPP2 and LPP3 are constitutively co-localized with SK1 (sphingosine kinase 1) in cytoplasmic vesicles in HEK-293 cells. Moreover, LPP2 but not LPP3 prevents SK1 from being recruited to a perinuclear compartment upon induction of PLD1 (phospholipase D1) in CHO (Chinese-hamster ovary) cells. Taken together, these data are consistent with an important role for LPP2 and LPP3 in regulating an intracellular pool of PA and S1P respectively, that may govern the apoptotic status of the cell upon serum deprivation.
Insights into the quaternary association of proteins through structure graphs: a case study of lectins
Authors: Brinda K. V. , Surolia Avadhesha , Vishveshwara Sarawathi
Published: September 2005

The unique three-dimensional structure of both monomeric and oligomeric proteins is encoded in their sequence. The biological functions of proteins are dependent on their tertiary and quaternary structures, and hence it is important to understand the determinants of quaternary association in proteins. Although a large number of investigations have been carried out in this direction, the underlying principles of protein oligomerization are yet to be completely understood. Recently, new insights into this problem have been gained from the analysis of structure graphs of proteins belonging to the legume lectin family. The legume lectins are an interesting family of proteins with very similar tertiary structures but varied quaternary structures. Hence they have become a very good model with which to analyse the role of primary structures in determining the modes of quaternary association. The present review summarizes the results of a legume lectin study as well as those obtained from a similar analysis carried out here on the animal lectins, namely galectins, pentraxins, calnexin, calreticulin and rhesus rotavirus Vp4 sialic-acid-binding domain. The lectin structure graphs have been used to obtain clusters of non-covalently interacting amino acid residues at the intersubunit interfaces. The present study, performed along with traditional sequence alignment methods, has provided the signature sequence motifs for different kinds of quaternary association seen in lectins. Furthermore, the network representation of the lectin oligomers has enabled us to detect the residues which make extensive interactions (‘hubs’) across the oligomeric interfaces that can be targetted for interface-destabilizing mutations. The present review also provides an overview of the methodology involved in representing oligomeric protein structures as connected networks of amino acid residues. Further, it illustrates the potential of such a representation in elucidating the structural determinants of protein–protein association in general and will be of significance to protein chemists and structural biologists.
Interaction of SP-A (surfactant protein A) with bacterial rough lipopolysaccharide (Re-LPS), and effects of SP-A on the binding of Re-LPS to CD14 and LPS-binding protein
Authors: García-Verdugo Ignacio , Sánchez-Barbero Fernando , Soldau Katrin , Tobias Peter S. , Casals Cristina
Published: September 2005

SP-A (surfactant protein A) is a lipid-binding collectin primarily involved in innate lung immunity. SP-A interacts with the bacterial rough LPS (lipopolysaccharide) Re-LPS (Re595 mutant of LPS from Salmonella minnesota), but not with smooth LPS. In the present study, we first examined the characteristics of the interaction of human SP-A with Re-LPS. Fluorescence intensity and anisotropy measurements of FITC-labelled Re-LPS in the presence and absence of SP-A indicated that SP-A bound to Re-LPS in solution in a Ca2+-independent manner, with a dissociation constant of 2.8×10−8 M. In the presence of calcium, a high-mobility complex of SP-A and [3H]Rb-LPS (Rb mutant of LPS from Escherichia coli strain LCD 25) micelles was formed, as detected by sucrose density gradients. Re-LPS aggregation induced by SP-A was further characterized by light scattering. On the other hand, human SP-A inhibited TNF-α (tumour necrosis factor-α) secretion by human macrophage-like U937 cells stimulated with either Re-LPS or smooth LPS. We further examined the effects of human SP-A on the binding of Re-LPS to LBP (LPS-binding protein) and CD14. SP-A decreased the binding of Re-LPS to CD14, but not to LBP, as detected by cross-linking experiments with 125I-ASD-Re-LPS [125I-labelled sulphosuccinimidyl-2-(p-azidosalicylamido)-1,3-dithiopropionate derivative of Re-LPS] and fluorescence analysis with FITC-Re-LPS. When SP-A, LBP and CD14 were incubated together, SP-A reduced the ability of LBP to transfer 125I-ASD-Re-LPS to CD14. These SP-A effects were not due to the ability of SP-A to aggregate Re-LPS in the presence of calcium, since they were observed in both the absence and the presence of calcium. These studies suggest that SP-A could contribute to modulate Re-LPS responses by altering the competence of the LBP–CD14 receptor complex.
An essential dimer-forming subregion of the endoplasmic reticulum stress sensor Ire1
Authors: Oikawa Daisuke , Kimata Yukio , Takeuchi Masato , Kohno Kenji
Published: September 2005

The luminal domain of the type I transmembrane protein Ire1 senses endoplasmic reticulum stress by an undefined mechanism to up-regulate the signalling pathway for the unfolded protein response. Previously, we proposed that the luminal domain of yeast Ire1 is divided into five subregions, termed subregions I–V sequentially from the N-terminus. Ire1 lost activity when internal deletions of subregion II or IV were made. In the present paper, we show that partial proteolysis of a recombinant protein consisting of the Ire1 luminal domain suggests that subregions II–IV are tightly folded. We also show that a recombinant protein of subregions II–IV formed homodimers, and that this homodimer formation was impaired by an internal deletion of subregion IV. Furthermore, recombinant fragments of subregion IV exhibited a self-binding ability. Therefore, although its sequence is little conserved evolutionarily, subregion IV plays an essential role to promote Ire1 dimer formation.
Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells
Authors: Zidi-Yahiaoui Nedjma , Mouro-Chanteloup Isabelle , D'Ambrosio Anne-Marie , Lopez Claude , Gane Pierre , Le Van Kim Caroline , Cartron Jean-Pierre , Colin Yves , Ripoche Pierre
Published: September 2005

The mammalian Rh (Rhesus) protein family belongs to the Amt/Mep (ammonia transporter/methylammonium permease)/Rh superfamily of ammonium transporters. Whereas RhCE, RhD and RhAG are erythroid specific, RhBG and RhCG are expressed in key organs associated with ammonium transport and metabolism. We have investigated the ammonium transport function of human RhBG and RhCG by comparing intracellular pH variation in wild-type and transfected HEK-293 (human embryonic kidney) cells and MDCK (Madin–Darby canine kidney) cells in the presence of ammonium (NH4+/NH3) gradients. Stopped-flow spectrofluorimetry analysis, using BCECF [2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein] as a pH-sensitive probe, revealed that all cells submitted to inwardly or outwardly directed ammonium gradients exhibited rapid alkalinization or acidification phases respectively, which account for ammonium movements in transfected and native cells. However, as compared with wild-type cells known to have high NH3 lipid permeability, RhBG- and RhCG-expressing cells exhibited ammonium transport characterized by: (i) a five to six times greater kinetic rate-constant; (ii) a weak temperature-dependence; and (iii) reversible inhibition by mercuric chloride (IC50: 52 μM). Similarly, when subjected to a methylammonium gradient, RhBG- and RhCG-expressing cells exhibited kinetic rate constants greater than those of native cells. However, these constants were five times higher for RhBG as compared with RhCG, suggesting a difference in substrate accessibility. These results, indicating that RhBG and RhCG facilitate rapid and low-energy-dependent bi-directional ammonium movement across the plasma membrane, favour the hypothesis that these Rh glycoproteins, together with their erythroid homologue RhAG [Ripoche, Bertrand, Gane, Birkenmeier, Colin and Cartron (2005) Proc. Natl. Acad. Sci. U.S.A. 101, 17222–17227] constitute a family of NH3 channels in mammalian cells.
Human ADA2 belongs to a new family of growth factors with adenosine deaminase activity
Authors: Zavialov Andrey V. , Engström Åke
Published: September 2005

Two distinct isoenzymes of ADA (adenosine deaminase), ADA1 and ADA2, have been found in humans. Inherited mutations in ADA1 result in SCID (severe combined immunodeficiency). This observation has led to extensive studies of the structure and function of this enzyme that have revealed an important role for it in lymphocyte activation. In contrast, the physiological role of ADA2 is unknown. ADA2 is found in negligible quantities in serum and may be produced by monocytes/macrophages. ADA2 activity in the serum is increased in various diseases in which monocyte/macrophage cells are activated. In the present study, we report that ADA2 is a heparin-binding protein. This allowed us to obtain a highly purified enzyme and to study its biochemistry. ADA2 was identified as a member of a new class of ADGFs (ADA-related growth factors), which is present in almost all organisms from flies to humans. Our results suggest that ADA2 may be active in sites of inflammation during hypoxia and in areas of tumour growth where the adenosine concentration is significantly elevated and the extracellular pH is acidic. Our finding that ADA2 co-purified and concentrated together with IgG in commercially available preparations offers an intriguing explanation for the observation that treatment with such preparations leads to non-specific immune-system stimulation.
A TrkA-to-p75NTR molecular switch activates amyloid β-peptide generation during aging
Authors: Costantini Claudio , Weindruch Richard , Della Valle Giuliano , Puglielli Luigi
Published: September 2005

Aging is the single most important risk factor for AD (Alzheimer's disease). However, the molecular events that connect normal aging to AD are mostly unknown. The abnormal accumulation of Aβ (amyloid β-peptide) in the form of senile plaques is one of the main characteristics of AD. In the present study, we show that two members of the neurotrophin receptor superfamily, TrkA (tyrosine kinase receptor A) and p75NTR (p75 neurotrophin receptor), differentially regulate the processing of APP (amyloid precursor protein): TrkA reduces, whereas p75NTR activates, β-cleavage of APP. The p75NTR-dependent effect requires NGF (nerve growth factor) binding and activation of the second messenger ceramide. We also show that normal aging activates Aβ generation in the brain by ‘switching’ from the TrkA to the p75NTR receptor system. Such an effect is abolished in p75NTR ‘knockout’ animals, and can be blocked by both caloric restriction and inhibitors of nSMase (neutral sphingomyelinase). In contrast with caloric restriction, which prevents the age-associated up-regulation of p75NTR expression, nSMase inhibitors block the activation of ceramide. When taken together, these results indicate that the p75NTR–ceramide signalling pathway activates the rate of Aβ generation in an age-dependent fashion, and provide a new target for both the understanding and the prevention of late-onset AD.
AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain
Authors: Mîinea Cristinel P. , Sano Hiroyuki , Kane Susan , Sano Eiko , Fukuda Mitsunori , Peränen Johan , Lane William S. , Lienhard Gustav E.
Published: September 2005

Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.
WNK lies upstream of kinases involved in regulation of ion transporters
Authors: Gamba Gerardo
Published: September 2005

Two members of a recently discovered family of protein kinases {WNK1 and WNK4 [with no K (lysine) kinases-1 and -4]} are the cause of an inherited disease known as pseudohypoaldosteronism type II that features arterial hypertension. The family is known as WNK due to a lack of the invariant catalytic lysine in kinase subdomain II. The mechanisms by which WNKs regulate blood pressure are beginning to be understood at the physiological level from recent studies showing effects of WNK4 on several plasma membrane co-transporters and ion channels. However, little is known about the function of WNKs at the biochemical level. In this issue of the Biochemical Journal, Vitari et al. have shown that WNK1 and WNK4 interact with other kinases, SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress response kinase-1), which are involved in the regulation of ion transporters. WNK1 and WNK4 phosphorylate SPAK and OSR1, which in turn phosphorylate the N-terminal domain of the basolateral Na+–K+–2Cl− co-transporter, NKCCl. The phosphorylation site involved in SPAK or OSR1 activation is identified as a threonine residue within the T-loop.
Serum levels of osteoprotegerin and RANKL in patients with ST elevation acute myocardial infarction
Authors: Crisafulli Alessandra , Micari Antonio , Altavilla Domenica , Saporito Francesco , Sardella Aurora , Passaniti Maria , Raffa Santi , D'Anneo Gaspare , Lucà Fabiana , Mioni Chiara , Arrigo Francesco , Squadrito Francesco
Published: September 2005

OPG (osteoprotegerin) has been suggested to have an important role in atherogenesis and vascular calcification. In the present study, we have investigated serum OPG and RANKL (receptor activator of nuclear factor κB ligand) concentrations in patients with ST elevation AMI (acute myocardial infarction) and established CAD (coronary artery disease). OPG and RANKL were measured in 58 male patients hospitalized in the coronary care unit with ST elevation AMI, in 52 asymptomatic male patients with an established diagnosis of CAD and in 52 healthy male controls. These last two groups were matched with the AMI patients for age and body mass index. OPG was significantly (P<0.05) higher in patients with AMI at 1 h after AMI (8.04±4.86 pmol/l) than in both patients with established CAD (4.92±1.65 pmol/l) and healthy subjects (3.15±1.01 pmol/l). Subjects with established CAD had significantly (P<0.05) increased OPG levels compared with controls. RANKL levels in patients with established CAD (0.02±0.05 pmol/l) and with AMI (0.11±0.4 pmol/l) were significantly (P<0.05) lower compared with controls (0.32±0.35 pmol/l). In the AMI group, OPG decreased significantly (P<0.05) at 1 and 4 weeks after infarction (8.04±4.86 compared with 6.38±3.87 and 6.55±2.6 pmol/l respectively), but OPG levels, either at 1 h or 1–4 weeks after AMI, remained significantly (P<0.05) higher compared with established CAD (4.92±1.65 pmol/l) and controls (3.15±1.01 pmol/l). Our data show for the first time that OPG levels are increased in ST elevation AMI within 1 h of infarction. Whether the increase in OPG is a consequence or a causal factor of plaque destabilization deserves further investigation.
Endothelial function and baroreflex sensitivity according to the oral glucose tolerance test in patients with coronary artery disease and normal fasting glucose levels
Authors: Wykretowicz Andrzej , Guzik Przemysław , Bartkowiak Grzegorz , Krauze Tomasz , Kąsinowski Ryszard , Dziarmaga Mieczysław , Wesseling Karel H. , Wysocki Henryk
Published: September 2005

Endothelial dysfunction and reduced BRS (baroreflex sensitivity) may be present in patients with CAD (coronary artery disease). The normal fasting glucose level does not exclude abnormal glucose metabolism in patients with CAD. The aim of present study was to evaluate endothelial function and BRS according to glucose metabolism in patients with normal fasting plasma glucose and stable CAD subjected to PTCA (percutaneous transluminal coronary angioplasty). Forty-six consecutive patients who underwent elective PTCA were studied (37 men; mean age 56 years). Endothelial function was assessed non-invasively using the arterial vasodilator response to salbutamol (albuterol). BRS was measured using a cross-correlation method. The extent of coronary narrowing was estimated by calculation of the Gensini score. All patients underwent a 75 g OGTT (oral glucose tolerance test). IGT (impaired glucose tolerance) or diabetes was present in approx. 60% of patients. The vasodilator response to salbutamol, as a measure of endothelial dysfunction, was significantly impaired in patients with IGT or diabetes compared with those with normal glucose tolerance (−0.5±1.6% compared with −7.9±2.2; P=0.01). Glucose metabolism and age were significant predictors of endothelial dysfunction (R2=35.2%, P=0.02). BRS did not differ significantly between patients with normal glucose tolerance and those with IGT or diabetes (6.9±1.2 compared with 6.1±0.6 ms/mmHg respectively; P=0.669). BRS was negatively correlated with age (r=−0.34, P=0.021) and the Gensini score (r=−0.34, P=0.022). The significant predictors of BRS were Gensini score, age and past myocardial infarction (R2=37.02%, P=0.002). Patients with established CAD, normal fasting glucose and IGT or diabetes demonstrated impaired endothelial function which did not correlate with the extent of coronary artery involvement. Conversely, BRS in the study population was not affected by glucose metabolism, but showed an interaction with the extent of coronary narrowing.
Equilibrium sampling of self-associating polymer solutions: A parallel selective tempering approach
Authors: Ayyagari Chakravarthy , Bedrov Dmitry , Smith Grant D.
Published: September 2005

We present a novel simulation algorithm based on tempering a fraction of relaxation-limiting interactions to accelerate the process of obtaining uncorrelated equilibrium configurations of self-associating polymer solutions. This approach consists of tempering (turning off) the attractive interactions for a fraction of self-associating groups determined by a biasing field h. A number of independent configurations (replicas) with overlapping Hamiltonian distributions in the expanded (NVTh) ensemble with constant NVT but different biasing fields, forming a chain of Hamiltonians, were simulated in parallel with occasional attempts to exchange the replicas associated with adjacent fields. Each field had an associated distribution of tempered interactions, average fraction of tempered interactions, and structural decorrelation time. Tempering parameters (number of replicas, fields, and exchange frequencies) were chosen to obtain the highest efficiency in sampling equilibrium configurations of a self-association polymer solution based on short serial simulation runs and a statistical model. Depending on the strength of the relaxation-limiting interactions, system size, and thermodynamic conditions, the algorithm can be orders of magnitude more efficient than conventional canonical simulation and is superior to conventional temperature parallel tempering.
The NaK 1Δ1,3 states: Theoretical and experimental studies of fine and hyperfine structure of rovibrational levels near the dissociation limit
Authors: Wilkins A. D. , Morgus L. , Hernandez-Guzman J. , Huennekens J. , Hickman A. P.
Published: September 2005

Earlier high-resolution spectroscopic studies of the fine and hyperfine structure of rovibrational levels of the 1Δ3 state of NaK have been extended to include high lying rovibrational levels with v⩽59, of which the highest levels lie within ∼4cm−1 of the dissociation limit. A potential curve is determined using the inverted perturbation approximation method that reproduces these levels to an accuracy of ∼0.026cm−1. For the largest values of v, the outer turning points occur near R∼12.7Å, which is sufficiently large to permit the estimation of the C6 coefficient for this state. The fine and hyperfine structure of the 1Δ3 rovibrational levels has been fit using the matrix diagonalization method that has been applied to other states of NaK, leading to values of the spin–orbit coupling constant Av and the Fermi contact constant bF. New values determined for v⩽33 are consistent with values determined by a simpler method and reported earlier. The measured fine and hyperfine structure for v in the range 44⩽v⩽49 exhibits anomalous behavior whose origin is believed to be the mixing between the 1Δ3 and 1Δ1 states. The matrix diagonalization method has been extended to treat this interaction, and the results provide an accurate representation of the complicated patterns that arise. The analysis leads to accurate values for Av and bF for all values of v⩽49. For higher v(50⩽v⩽59), several rovibrational levels have been assigned, but the pattern of fine and hyperfine structure is difficult to interpret. Some of the observed features may arise from effects not included in the current model.