bluuebunny/crossref_metadata_2025 huggingface dataset:
SOME LESSONS ON CONSTITUTION-MAKING FROM ZIMBABWE
Authors: Hatchard John
Published: April 2005

The former (and late lamented) Chief Justice of South Africa, Justice Ismail
Mohammed, once observed that:

“The constitution of a nation is not simply a statute which mechanically defines
the structures of government and the relations between the government and the
governed, it is a ‘mirror of the national soul’, the identification of the ideals and
aspirations of a nation, the articulation of the values binding its people and
disciplining its government.”

Further, as van der Vyer has warned, “. . . a superimposed constitutional
formulae or constitutional arrangements that . . . do not address the real causes
of discontent, are sure to generate their own legitimacy crisis.” It follows that
the development of an appropriate procedure for constitution-making is of the
greatest practical importance. This short comment seeks to examine critically
the much-publicized efforts in Zimbabwe to develop a new autochthonous
constitution and to draw some lessons therefrom.
CASE NOTE
Authors: Osode Patrick C.
Published: April 2005

DEFINING THE LIMITS OF PERMISSIBLE EMPLOYMENT DISCRIMINATION AGAINST PERSONS LIVING WITH HIV/AIDS IN SOUTH AFRICA: HOFFMAN V. SOUTH AFRICAN AIRWAYS [2000] 12 BLLR 1365.

Perhaps the most positive and exciting aftermath of the apartheid era is the
construction of the new South Africa upon the foundation of a Constitution
and other legal instruments that are unanimous and unambiguous in two
respects. The first is in their proscription of unfair discrimination and the second
is in their permission of statutory and other measures aimed at eliminating the
effects of past discrimination on those groups of persons who were at the receiving
end of same. The provisions of these instruments as well as their tenor and
spirit reveal an unmistakable national resolve to break from a culture of racial
discrimination to a constitutionally protected culture of human rights for South
Africans of all ages, classes and colours. Without doubt, the most important of
those provisions is the equality clause of the Bill of Rights contained in the
second chapter of the Constitution. This probably follows from a realization of
the fact that equality is fundamental to “the maintenance and propagation of
human rights in a democratic body politic, particularly in an acutely divided
society” such as South Africa.
Conflicts of interest in biofilms
Authors: Kreft J.-U.
Published: April 2005

High cell density and close proximity of diverse species of microorganisms are typical of life in natural biofilms. These conditions give ample opportunity for both competitive and cooperative interactions between individuals of the same and different species. Cooperative behaviour benefits the group of neighbouring microbes but comes at a fitness cost for the cooperating individuals. This creates a conflict of interest between the fitness of the individual and the fitness of the group. Individuals that defect from cooperation and therefore do not pay the cost but nevertheless benefit from the cooperative behaviour of others are called cheaters. Cooperative behaviour in the presence of cheaters constitutes altruism towards the cheaters. The aim of this review is two-fold: first, to introduce key concepts from kin selection and group selection theory that allow us to understand how cooperative behaviour can evolve in the face of cheaters; secondly, to draw attention to the conflicts of interest prevalent in biofilms yet largely ignored in the biofilm literature. Examples discussed comprise growth restraint in stationary phase as an instance of the Prisoner's Dilemma, growth restraint to allow channel formation, restraint in resource consumption or economical use of resources as altruistic behaviour, population heterogeneity as insurance against environmental changes, cooperative investment in diffusible exoenzymes, cooperation of pathogens and virulence, diffusion sensing versus quorum sensing and the inflation of signals, antibiotic resistance as collective action, and programmed cell death.
Early events and pattern formation in Listeria monocytogenes biofilms
Authors: Takhistov P. , George B.
Published: April 2005

Biofilm initiation and development is a complex process that includes several major stages. This study examined the process of Listeria monocytogenes biofilm initiation and consequent spatial and physiological patterns formed in the biofilm as the result of bacterial response to a changing environment. This is a first attempt at establishing a link between the spatial organization of biofilm-associated bacteria and major stress factors such as surface bioavailability and nutrient content. Developed linear models allow evaluation of some important parameters of the surface population, such as the critical colony size and the intercolony distance in the case of diffusion-limited nutrient access. Variations in bacterial physiological patterns during biofilm initiation as a function of bacterial population density during surface colonization are reported.
Modeling biofilm complexity by including active and inert biomass and extracellular polymeric substances
Authors: Laspidou C. S. , Rittmann B. E.
Published: April 2005

We present an overview of all the features of the unified multi-component cellular automaton (UMCCA) model. The UMCCA model describes quantitatively the complexity of biofilms for all biofilm components: active bacteria, inert biomass and extracellular polymeric substances (EPS). It also includes original donor substrate, two types of soluble microbial products (SMP) and oxygen. All mass balances and parameters used in UMCCA are based on our unified theory, which reconciles the apparently disparate findings about active and inert biomass, EPS and SMP. The UMCCA model captures all trends observed experimentally regarding biofilm density, namely large density differences within individual biofilm samples, with bottom layers (closest to the substratum) being as much as 5 to 10 times denser than top layers, which appear to be “fluffy” and porous. To capture this effect, the UMCCA model employs the novel idea of biofilm consolidation: the fluid over the biofilm creates pressures and vibrations that cause the biofilm to consolidate, or pack itself to a higher density over time. As a result, each biofilm compartment in the model output consolidates to a different degree that depends on the age of its biomass, which is higher for the bottom layers of the biofilm. The UMCCA model can also be used to describe biofilm mechanical properties variable in time and space, and it can be linked to finite-element software to study the behavior of biofilms under tensile or compressive stress and to perform a stress analysis of the deformed biofilm, possibly indicating where it is likely to fail or detach.
Introduction to Proceedings of the Workshop “Biocomplexity VI: Complex Behavior in Unicellular Organisms”
Authors: Fuqua C. , Glazier J. A. , Brun Y. , Alber M. S.
Published: April 2005

This special issue of Biofilms highlights current experimental studies and modeling of collective phenomena in single-celled microbes, primarily bacteria, stemming from the Workshop “Biocomplexity VI: Complex Behavior in Unicellular Organisms”, held 12–16 May 2004, at Indiana University, Bloomington, IN, USA, and co-organized by the Biocomplexity Institute at Indiana University and the Interdisciplinary Center for the Study of Biocomplexity at the University of Nôtre Dame, with support from the National Science Foundation (grant no. 0352904), the National Institutes of Health (grant no. GM071709-01), the National Institute of General Medical Sciences and the College of Arts and Sciences at Indiana University.
Bedeutung und Ergebnisse der Theory of Mind-Forschung für den Autismus und andere psychiatrische Erkrankungen
Authors: Bruning Nicole , Konrad Kerstin , Herpertz-Dahlmann Beate
Published: April 2005

Zusammenfassung: Erkrankungen des autistischen Spektrums sind im Wesentlichen durch folgende Symptomtriade gekennzeichnet: Störung der sozialen Interaktion, Störung der Kommunikation, sowie stereotype Verhaltensmuster und Interessen. Einer der prominentesten Ansätze zur Erklärung dieser Auffälligkeiten stellt die «Theory of Mind» dar ( Baron-Cohen et al., 1985 ). Die vorliegende Übersichtsarbeit diskutiert den aktuellen Forschungsstand hierzu und stellt wesentliche Ergebnisse der letzten 10 Jahre zusammenfassend dar. Gleichzeitig werden diese kritisch hinsichtlich ihrer Bedeutsamkeit für die Erklärung anderer psychiatrischer Erkrankungen und ihrer methodischen Qualität betrachtet. Trotz der vielfältigen Forschungsbemühungen zu diesem Thema stellen die unterschiedlichen Störungskonzepte noch kein einheitliches Rahmenmodell zur Entstehung der Erkrankungen des autistischen Spektrums dar. Zukünftige Forschung sollte sich zum Ziel setzen, den Erklärungswert der einzelnen theoretischen Modelle genauer zu überprüfen und Gemeinsamkeiten der verschiedenen Konzepte herauszustellen, um so gezieltere Informationen über potentielle Verursachungsfaktoren von Autismus zu bekommen.
A pervasive role of ubiquitin conjugation in activation and termination of IκB kinase pathways
Authors: Krappmann Daniel , Scheidereit Claus
Published: April 2005

The nuclear factor (NF)‐κB pathway is a paradigm for gene expression control by ubiquitin‐mediated protein degradation. In stimulated cells, phosphorylation by the IκB kinase (IKK) complex primes NF‐κB‐inhibiting IκB molecules for lysine (Lys)‐48‐linked polyubiquitination and subsequent destruction by the 26S proteasome. However, recent studies indicate that the ubiquitin (Ub) system controls NF‐κB pathways at many levels. Ub ligases are activated by different upstream signalling pathways, and they function as central regulators of IKK and c‐Jun amino‐terminal kinase activation. The assembly of Lys 63 polyUb chains provides docking surfaces for the recruitment of IKK‐activating complexes, a reaction that is counteracted by deubiquitinating enzymes. Furthermore, Ub conjugation targets upstream signalling mediators as well as nuclear NF‐κB for post‐inductive degradation to limit the duration of signalling.
Homotypic dimerization of the actin-binding protein p57/coronin-1 mediated by a leucine zipper motif in the C-terminal region
Authors: OKU Teruaki , ITOH Saotomo , ISHII Rie , SUZUKI Kensuke , NAUSEEF William M. , TOYOSHIMA Satoshi , TSUJI Tsutomu
Published: April 2005

The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in immune cells, and has been implicated in leucocyte migration and phagocytosis by virtue of its interaction with F-actin (filamentous actin). We previously identified two sites in the N-terminal region of p57/coronin-1 by which it binds actin, and in the present study we examine the role of the leucine zipper motif located in the C-terminal coiled-coil domain in mediating the homotypic association of p57/coronin-1. Recombinant p57/coronin-1 protein in solution formed a homodimer, as analysed by Superose 12 column chromatography and by sucrose density gradient centrifugation. In vivo, a truncated form consisting of the C-terminal coiled-coil domain co-precipitated with full-length p57/coronin-1 when both were co-expressed in COS-1 cells. A chimaeric construct composed of the C-terminal domain of p57/coronin-1 (which lacks the actin-binding sites) fused with green fluorescent protein co-localized with cortical F-actin-rich regions in COS-1 cells only when full-length p57/coronin-1 was expressed simultaneously in the cells, suggesting that the C-terminal region is required for the homotypic association of p57/coronin-1. Furthermore, p57LZ, a polypeptide consisting of the C-terminal 90 amino acid residues of p57/coronin-1, was sufficient for dimerization. When two leucine residues out of the four that constitute the leucine zipper structure in p57LZ or full-length p57 were replaced with alanine residues, the mutants failed to form homodimers. Taken together, these results demonstrate that p57/coronin-1 forms homodimers, that the association is mediated by the leucine zipper structure in the C-terminal region, and that it plays a role in the cross-linking of F-actin in the cell.
Characterization of phylogenetically distant members of the adenylate cyclase family from mycobacteria: Rv1647 fromMycobacterium tuberculosisand its orthologue ML1399 fromM. leprae
Authors: SHENOY Avinash R. , SREENATH Nandini P. , MAHALINGAM Mohana , VISWESWARIAH Sandhya S.
Published: April 2005

Analysis of the genome sequence of Mycobacterium tuberculosis H37Rv has identified 16 genes that are similar to the mammalian adenylate and guanylate cyclases. Rv1647 was predicted to be an active adenylate cyclase but its position in a phylogenetically distant branch from the other enzymes characterized so far from M. tuberculosis makes it an interestingly divergent nucleotide cyclase to study. In agreement with its divergence at the sequence level from other nucleotide cyclases, the cloning, expression and purification of Rv1647 revealed differences in its biochemical properties from the previously characterized Rv1625c adenylate cyclase. Adenylate cyclase activity of Rv1647 was activated by detergents but was resistant to high concentrations of salt. Mutations of substrate-specifying residues to those present in guanylate cyclases failed to convert the enzyme into a guanylate cyclase, and did not alter its oligomeric status. Orthologues of Rv1647 could be found in M. leprae, M. avium and M. smegmatis. The orthologue from M. leprae (ML1399) was cloned, and the protein was expressed, purified and shown biochemically to be an adenylate cyclase, thus representing the first adenylate cyclase to be described from M. leprae. Importantly, Western-blot analysis of subcellular fractions from M. tuberculosis and M. leprae revealed that the Rv1647 and ML1399 gene products respectively were expressed in these bacteria. Additionally, M. tuberculosis was also found to express the Rv1625c adenylate cyclase, suggesting that multiple adenylate cyclase proteins may be expressed simultaneously in this organism. These results suggest that class III cyclase-like gene products probably have an important role to play in the physiology and perhaps the pathology of these medically important bacteria.
TraA and its N-terminal relaxase domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as dimers in solution
Authors: KOPEC Jolanta , BERGMANN Alexander , FRITZ Gerhard , GROHMANN Elisabeth , KELLER Walter
Published: April 2005

TraA is the DNA relaxase encoded by the broad-host-range Grampositive plasmid pIP501. It is the second relaxase to be characterized from plasmids originating from Gram-positive organisms. Full-length TraA (654 amino acids) and the N-terminal domain (246 amino acids), termed TraAN246, were expressed as 6×His-tagged fusions and purified. Small-angle X-ray scattering and chemical cross-linking proved that TraAN246 and TraA form dimers in solution. Both proteins revealed oriTpIP501 (origin of transfer of pIP501) cleavage activity on supercoiled plasmid DNA in vitro. oriT binding was demonstrated by electrophoretic mobility shift assays. Radiolabelled oligonucleotides covering different parts of oriTpIP501 were subjected to binding with TraA and TraAN246. The KD of the protein–DNA complex encompassing the inverted repeat, the nick site and an additional 7 bases was found to be 55 nM for TraA and 26 nM for TraAN246. The unfolding of both protein constructs was monitored by measuring the change in the CD signal at 220 nm upon temperature change. The unfolding transition of both proteins occurred at approx. 42 °C. CD spectra measured at 20 °C showed 30% α-helix and 13% β-sheet for TraA, and 27% α-helix and 18% β-sheet content for the truncated protein. Upon DNA binding, an enhanced secondary structure content and increased thermal stability were observed for the TraAN246 protein, suggesting an induced-fit mechanism for the formation of the specific relaxase–oriT complex.
Regulation of activity in vitro and in vivo of three phospholipases B from Saccharomyces cerevisiae
Authors: MERKEL Olaf , OSKOLKOVA Olga V. , RAAB Florian , EL-TOUKHY Rosemarie , PALTAUF Fritz
Published: April 2005

The genome of the yeast, Saccharomyces cerevisiae, contains three highly similar genes coding for phospholipases B/lysophospholipases. These enzymes behave differently with respect to substrate preferences in vitro and relative contributions to phospholipid catabolism in vivo [Merkel, Fido, Mayr, Prüger, Raab, Zandonella, Kohlwein and Paltauf (1999) J. Biol. Chem. 274, 28121–28127]. It is shown in the present study that, in vitro, pH markedly affects the substrate preference of Plb1p and Plb2p, but not of Plb3p. At the pH optimum of 2.5–3.5, the order of substrate preference of Plb1p and Plb2p is PtdSer (phosphatidylserine)>PtdIns>PtdCho (phosphatidylcholine>PtdEtn (phosphatidylethanolamine). At pH values of 5 and above, the substrate preferences change to PtdCho=PtdEtn for Plb1p and PtdSer=PtdEtn for Plb2p. Accordingly, with cultured cells the ratio of PtdIns/PtdCho breakdown, as reflected in the ratio of GroPIns (glycerophosphoinositol)/GroPCho (glycerophosphocholine) released into the culture medium, is inversely related to the pH of the growth medium. This effect is ascribed to the pH response of Plb1p, because Plb2p does not contribute to the degradation of PtdIns and PtdCho in vivo. Bivalent and tervalent cations activate phospholipases B at pH 5.5, but are inhibitory at pH 2.5. Al3+ at a concentration of 20 mM increases Plb1p activity in vitro by 8-fold and leads to a 9-fold increase in GroPCho release by whole cells. In vivo, cycloheximide strongly inhibits the breakdown of PtdIns, and to a lesser extent PtdCho. However, Al3+-stimulated GroPCho release is almost completely inhibited by cycloheximide. Deletion of PLB3 leads to increased sensitivity to toxic Al3+. Addition of SDS or melittin to cultured cells leads to a significant increase in phospholipid degradation, which is insensitive to inhibition by cycloheximide. Deletion mutants defective in the PLB1 gene are significantly more resistant to SDS than are wild-type cells.
Depletion of Caco-2 cell cholesterol disrupts barrier function by altering the detergent solubility and distribution of specific tight-junction proteins
Authors: LAMBERT Daniel , O'NEILL Catherine A. , PADFIELD Philip J.
Published: April 2005

In the present study, we have investigated the role of cholesterol in maintaining the barrier properties of the model intestinal cell line Caco-2. We have extracted membrane cholesterol using methyl-β-cyclodextrin and demonstrated that maximally, methyl-β-cyclodextrin lowered cell cholesterol levels by 40–45%. Depletion of cell cholesterol was accompanied by an 80–90% decrease in monolayer transepithelial electrical resistance and a significant increase in the paracellular permeability of dextrans of 4, 10 and 40 kDa. The increase in dextran permeability was most pronounced for the two lower molecular mass species. In addition to the decline in the barrier properties of the monolayers, extraction of cell cholesterol produced an increase in the Triton X-100 solubility of claudin 3, claudin 4 and occludin, and the loss of all three proteins from the plasma membrane (tight junctions). In contrast, removal of cholesterol had no detectable influence on the detergent solubility or morphological distribution of claudin 1. These results indicate that membrane cholesterol is a critical factor in maintaining the barrier property of epithelial monolayers. More specifically, cholesterol appears to stabilize the association of certain proteins with the tight junctions.
Shedding and γ-secretase-mediated intramembrane proteolysis of the mucin-type molecule CD43
Authors: ANDERSSON Christian X. , FERNANDEZ-RODRIGUEZ Julia , LAOS Sirle , BAECKSTRÖM Dan , HAASS Christian , HANSSON Gunnar C.
Published: April 2005

CD43 is a transmembrane molecule that contains a 123-aminoacids-long cytoplasmic tail and a highly O-glycosylated extracellular domain of mucin type. Endogenous CD43 expressed in COLO 205, K562 and Jurkat cells revealed a membrane-associated, 20 kDa CD43-specific cytoplasmic tail fragment (CD43-CTF) upon inhibition of γ-secretase. This fragment was formed by an extracellular cleavage, as it was not accumulated after treating cells with 1,10-phenanthroline, a metalloprotease inhibitor. When CD43 was transfected into HEK-293 cells expressing dominant-negative PS1 (presenilin-1), the CD43-CTF was accumulated, but not in cells with wild-type PS1. Owing to its accumulation in the presence of a non-functional PS variant, it may thus be a novel γ-secretase substrate. This CTF is formed by an extracellular cleavage close to the membrane, is a fragment that can be concluded to be a substrate for γ-secretase. However, the intracellular γ-secretase product has not been possible to detect, suggesting a quick processing of this product. During normal growth the CTF was not found without γ-secretase inhibition, but when the cells (COLO 205) were very confluent the fragment could be detected. The intracellular domain of CD43 has previously been shown to contain a functional nuclear localization signal, and has been suggested to be involved in gene activation. From this and the present results, a novel way to explain how mucin-type molecules may transduce intracellular signals can be proposed.
The C-terminal domain of the HIV-1 regulatory protein Vpr adopts an antiparallel dimeric structure in solution via its leucine-zipper-like domain
Authors: BOURBIGOT Sarah , BELTZ Hervé , DENIS Jérôme , MORELLET Nelly , ROQUES Bernard P. , MÉLY Yves , BOUAZIZ Serge
Published: April 2005

HIV-1 Vpr is a highly conserved accessory protein that is involved in many functions of the virus life cycle. Vpr facilitates the entry of the HIV pre-integration complex through the nuclear pore, induces G2 cell cycle arrest, regulates cell apoptosis, increases transcription from the long terminal repeat and enhances viral replication. Vpr contains a Leu/Ile-rich domain (amino acids 60–81) in its C-terminal part, which is critical for dimerization. The sequence comprising residues 52–96 is implicated in properties of the protein such as DNA interaction and apoptosis via interaction with the adenine nucleotide translocator. To understand the specific interactions of Vpr-(52–96), the ability of this peptide to dimerize via a leucine-zipper mechanism has been investigated, by NMR and fluorescence spectroscopy. In contrast with results from a study performed in the presence of trifluoroethanol, our results, obtained in 30% (v/v) [2H]acetonitrile, show that Vpr-(52–96) in solution still forms an α-helix spanning residues 53–75, but dimerizes in an antiparallel orientation, through hydrophobic interactions between leucine and isoleucine residues and stacking between His71 and Trp54. Moreover, to demonstrate the physiological relevance of the dimer structure, fluorescence spectroscopy experiments have been performed in a Mes buffer, which confirmed the formation of the dimer in aqueous solution and highlighted the spatial proximity between Trp54 and His71. Surprisingly, the leucine-zipper structure shown in the present work for Vpr-(52–96) mimics the structure of full-length Vpr-(1–96), and this could explain why some of the properties of Vpr-(52–96) and Vpr-(1–96) are identical, while some are even enhanced for Vpr-(52–96), particularly in the case of DNA transfection experiments.
Characterization of the inositol phosphorylceramide synthase activity from Trypanosoma cruzi
Authors: FIGUEIREDO Juliana M. , DIAS Wagner B. , MENDONÇA-PREVIATO Lucia , PREVIATO José O. , HEISE Norton
Published: April 2005

IPC (inositol phosphorylceramide) synthase is an enzyme essential for fungal viability, and it is the target of potent antifungal compounds such as rustmicin and aureobasidin A. Similar to fungi and some other lower eukaryotes, the protozoan parasite Trypanosoma cruzi is capable of synthesizing free or protein-linked glycoinositolphospholipids containing IPC. As a first step towards understanding the importance and mechanism of IPC synthesis in T. cruzi, we investigated the effects of rustmicin and aureobasidin A on the proliferation of different life-cycle stages of the parasite. The compounds did not interfere with the axenic growth of epimastigotes, but aureobasidin A decreased the release of trypomastigotes from infected murine peritoneal macrophages and the number of intracellular amastigotes in a dose-dependent manner. We have demonstrated for the first time that all forms of T. cruzi express an IPC synthase activity that is capable of transferring inositol phosphate from phosphatidylinositol to the C-1 hydroxy group of C6-NBD-cer {6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]hexanoylceramide} to form inositol phosphoryl-C6-NBD-cer, which was purified and characterized by its chromatographic behaviour on TLC and HPLC, sensitivity to phosphatidylinositol-specific phospholipase C and resistance to mild alkaline hydrolysis. Unlike the Saccharomyces cerevisiae IPC synthase, the T. cruzi enzyme is stimulated by Triton X-100 but not by bivalent cations, CHAPS or fatty-acid-free BSA, and it is not inhibited by rustmicin or aureobasidin A, or the two in combination. Further studies showed that aureobasidin A has effects on macrophages independent of the infecting T. cruzi cells. These results suggest that T. cruzi synthesizes its own IPC, but by a mechanism that is not affected by rustmicin and aureobasidin A.
Molecular mousetraps and the serpinopathies
Authors: Lomas D.A. , Belorgey D. , Mallya M. , Miranda E. , Kinghorn K.J. , Sharp L.K. , Phillips R.L. , Page R. , Robertson A.S. , Crowther D.C.
Published: April 2005

Members of the serine proteinase inhibitor or serpin superfamily inhibit their target proteinases by a remarkable conformational transition that involves the enzyme being translocated more than 70 Å (1 Å=10−10 m) from the upper to the lower pole of the inhibitor. This elegant mechanism is subverted by point mutations to form ordered polymers that are retained within the endoplasmic reticulum of secretory cells. The accumulation of polymers underlies the retention of mutants of α1-antitrypsin and neuroserpin within hepatocytes and neurons to cause cirrhosis and dementia respectively. The formation of polymers results in the failure to secrete mutants of other members of the serpin superfamily: antithrombin, C1 inhibitor and α1-antichymotrypsin, to cause a plasma deficiency that results in the clinical syndromes of thrombosis, angio-oedema and emphysema respectively. Understanding the common mechanism underlying the retention and deficiency of mutants of the serpins has allowed us to group these conditions as the serpinopathies. We review in this paper the molecular and structural basis of the serpinopathies and show how this has allowed the development of specific agents to block the polymerization that underlies disease.
Therapeutic challenges of kinase and phosphatase inhibition and use in anti-diabetic strategy
Authors: Bridges A.J.
Published: April 2005

The development of kinase and phosphatase inhibitors as novel therapeutic agents has been stimulated by the discovery that most biological processes are controlled by the reversible phosphorylation of proteins. Most of the early results in this area were generated in oncology, at the same time as the human genome, with its 500+ kinases and 100+ phosphatases was deciphered. Because of this, we know a great deal about which processes signalling inhibitors interfere with, but little about the overall consequences. In this study, kinases will be briefly reviewed, followed by some of the early problems in developing kinase inhibitors, as biochemical reagents, and clinically active pharmaceuticals in oncology. The discussion will then switch to the potential role of kinases and phosphatases in controlling the disease process in Type II diabetes. Phosphatase inhibitors should augment insulin receptor tyrosine kinase signalling. Glycogen synthesis and glycogenolysis are phosphorylation dependent, and amenable to kinase inhibition, as are some nuclear hormone receptors, and these will be briefly discussed.
Role of protein kinase B in insulin-regulated glucose uptake
Authors: Welsh G.I. , Hers I. , Berwick D.C. , Dell G. , Wherlock M. , Birkin R. , Leney S. , Tavaré J.M.
Published: April 2005

The activation of protein kinase B (or Akt) plays a central role in the stimulation of glucose uptake by insulin. Currently, however, numerous questions remain unanswered regarding the role of this kinase in bringing about this effect. For example, we do not know precisely where in the GLUT4 trafficking pathway this kinase acts. Nor do we know which protein substrates are responsible for mediating the effects of protein kinase B, although two recently identified proteins (AS160 and PIKfyve) may play a role. This paper addresses these important questions by reviewing recent progress in the field.
Insulin signal transduction in human skeletal muscle: identifying the defects in Type II diabetes
Authors: Björnholm M. , Zierath J.R.
Published: April 2005

Type II diabetes is characterized by defects in insulin action on peripheral tissues, such as skeletal muscle, adipose tissue and liver and pancreatic β-cell defects. Since the skeletal muscle accounts for approx. 75% of whole body insulin-stimulated glucose uptake, defects in this tissue play a major role in the impaired glucose homoeostasis in Type II diabetic patients. Thus identifying defective steps in this process may reveal attractive targets for drug development to combat insulin resistance and Type II diabetes. This review will describe the effects of insulin on glucose transport and other metabolic events in skeletal muscle that are mediated by intracellular signalling cascades. Evidence for impaired activation of the insulin receptor signalling cascade and defective glucose transporter 4 translocation in the skeletal muscle from Type II diabetic patients will be presented. Through the identification of the intracellular defects in insulin action that control glucose homoeostasis, a better understanding of the disease pathogenesis can be gained and strategies for intervention may be developed.
Modulation of insulin signalling by insulin sensitizers
Authors: Jiang G. , Zhang B.B.
Published: April 2005

Insulin resistance is a hallmark of Type II diabetes. It is well documented that insulin sensitizers such as peroxisome-proliferator-activated receptor γ agonists and aspirin improve insulin action in vivo. The detailed mechanisms by which the insulin sensitizers promote insulin signalling, however, are not completely understood and remain somewhat controversial. In the present review, we summarize our studies attempting to explore the molecular mechanisms underlying the effects of insulin sensitizers in cells and in animal models of insulin resistance. In 3T3-L1 adipocytes and/or in HEK-293 cells stably expressing recombinant IRS1 protein (insulin receptor substrate protein 1), the peroxisome-proliferator-activated receptor γ agonist rosiglitazone and aspirin promote insulin signalling by decreasing inhibitory IRS1 serine phosphorylation. Increased IRS1 Ser-307 phosphorylation and concomitant decreased insulin signalling as measured by insulin-stimulated IRS1 tyrosine phosphorylation and Akt threonine phosphorylation were observed in adipose tissues of Zucker obese rats compared with lean control rats. Treatment with rosiglitazone for 24 and 48 h increased insulin signalling and decreased IRS1 Ser-307 phosphorylation concomitantly. Treatment of the Zucker obese rats with rosiglitazone for 24 h also reversed the high circulating levels of free fatty acids, which have been shown to correlate with increased IRS1 serine phosphorylation. Taken together, the results suggest that IRS1 inhibitory serine phosphorylation is a key component of insulin resistance and its reversal may be physiologically relevant to insulin sensitization in vivo.
Small molecule glucokinase activators as novel anti-diabetic agents
Authors: Leighton B. , Atkinson A. , Coghlan M.P.
Published: April 2005

The monomeric enzyme GK (glucokinase) has a low affinity for glucose and, quantitatively, is largely expressed in the liver and pancreatic β-cells, playing a key ‘glucose sensing’ role to regulate hepatic glucose balance and insulin secretion. Mutations of GK in man can be inactivating, to cause a form of diabetes mellitus, or activating, to lower blood glucose levels. Recently, models of GK protein structure have helped to elucidate the role of inactivating and activating mutations, with the latter revealing an allosteric binding site, possibly for an unknown physiological activator. However, this discovery was pre-dated by Drug Discovery projects that have identified small organic molecules that activate pancreatic and liver GK enzyme activity. These compounds stimulate insulin secretion in islets and glucose metabolism in hepatocytes. The profile of these GK activators, both in vitro and in vivo and the potential role that GK activators play in lowering blood glucose levels in Type II diabetes mellitus will be discussed.
Before and beyond ABA: upstream sensing and internal signals that determine ABA accumulation and response under abiotic stress
Authors: Verslues P.E. , Zhu J.-K.
Published: April 2005

Sensing and signalling events that detect abiotic stress-induced changes in plant water status and initiate downstream stress responses such as ABA (abscisic acid) accumulation and osmoregulation remain uncharacterized in plants. Although conclusive results are lacking, recent results from plants, and analogies to signalling in other organisms, suggest possible mechanisms for sensing altered water status and initial transduction of that signal. Internal signals that act downstream of ABA and modulate stress responses to reflect the type and severity of the stress and the metabolic status of the plant are also not well understood. Two specific types of signalling, sugar sensing and reactive oxygen signalling, are likely to be modulators of ABA response under stress. For both upstream sensing and signalling of plant water status as well as downstream modulation of ABA response, present results suggest several genetic strategies with high potential to increase our understanding of the molecular basis by which plants sense and respond to altered water status.
Post-translational regulation in plants employing a diverse set of polypeptide tags
Authors: Downes B. , Vierstra R.D.
Published: April 2005

The concept that plants exploit polypeptides as post-translational modifiers is rapidly emerging as an important method to manipulate various cellular processes. The best known is Ub (ubiquitin) that serves as reusable tag for selective protein degradation by the 26 S proteasome and for endosomal trafficking. Genomic analyses indicate that Ub pathway alone comprises over 6% of the Arabidopsis proteome with thousands of proteins being targets. Consequently, this pathway influences much of plant biology. Others tags include RUB-1 (related to Ub-1; also known as NEDD8), SUMO (small Ub-like modifier), ATG-8 (autophagy-8) and ATG-12, UFM-1 (Ub-fold modifier-1) and HUB-1 (homology to Ub-1). Preliminary studies indicate that these tags have much more limited sets of targets and provide more specialized functions, including transcriptional regulation, protein localization, autophagic turnover and antagonizing the effects of Ub. On the basis of their widespread distribution and pervasive functions, peptide tags can now be considered as prime players in plant cell regulation.
Coagulation inhibitors in inflammation
Authors: Esmon C.T.
Published: April 2005

Coagulation is triggered by inflammatory mediators in a number of ways. However, to prevent unwanted clot formation, several natural anticoagulant mechanisms exist, such as the antithrombin–heparin mechanism, the tissue factor pathway inhibitor mechanism and the protein C anticoagulant pathway. This review examines the ways in which these pathways are down-regulated by inflammation, thus limiting clot formation and decreasing the natural anti-inflammatory mechanisms that these pathways possess.
Harnessing wound healing and regeneration for tissue engineering
Authors: Metcalfe A.D. , Ferguson M.W.J.
Published: April 2005

Biomedical science has made major advances in understanding how cells grow into functioning tissue and the signalling mechanisms used to achieve this are slowly being dissected. Tissue engineering is the application of that knowledge to the building or repairing of organs, including skin, the largest organ in the body. Generally, engineered tissue is a combination of living cells and a supporting matrix. Besides serving as burn coverings, engineered skin substitutes can help patients with diabetic foot ulcers. Today, most of these ulcers are treated with an approach that includes antibiotics, glucose control, special shoes and frequent cleaning and bandaging. The results of such treatments are often disappointing and ineffectual, and scarring remains a major problem, mechanically, cosmetically and psychologically. Within our group we are attempting to address this by investigating novel approaches to skin tissue engineering. We are identifying novel therapeutic manipulations to improve the degree of integration between a tissue engineered dermal construct and the host by both molecular manipulation of growth factors but also by understanding and harnessing mechanisms of regenerative biology. For the purpose of this summary, we will concentrate primarily on the latter of these two approaches in that we have identified a novel mouse mutant that completely and perfectly regenerates skin and cartilaginous components following ear injury. This experimental animal will allow us to characterize not only novel genes involved in the regeneration process but also to utilize cells from such animals in artificial skin equivalents to assess their behaviour compared with normal cells. This approach should allow us to create a tissue-engineered substitute, which more closely resembles the normal regional microanatomy and physiology of the skin, allowing better integration to the host with minimal or no scarring.
The role of blood cells and their microparticles in blood coagulation
Authors: Eilertsen K.-E. , Østerud B.
Published: April 2005

The transmembrane glycoprotein TF (tissue factor) plays an essential role in haemostasis as the principal initiator of blood coagulation. In this paper, we describe how the circulating blood cells – monocytes, platelets, neutrophils and their microparticles – co-operate in regulating the expression, availability and activity of monocyte-derived TF.
Direct current electrical characterization of ds-DNA in nanogap junctions
Authors: Iqbal Samir M. , Balasundaram G. , Ghosh Subhasis , Bergstrom D. E. , Bashir R.
Published: April 2005

Measurements of DNA conductivity, hybridization, and melting using electronic means can have wide applications in molecular electronics and biological sensors. We have fabricated nanogap break-junctions by electromigration through thin gold-on-titanium films. 18-mer thiolated ds-DNA molecules were covalently attached between the electrodes and dc electrical measurements were done. The conductance was measured through the molecule before and after a temperature ramp from 300 to 400 K. A dramatic decrease in conductance was observed, analogous to an electrical fuse, possibly attributed to complete or partial denaturing of the ds-DNA molecules bridging the nanogaps. We also show evidence that the dc resistance of dry DNA strands of the same length decreases with increasing guanine-cytosine content in the sequence with values ranging from 10 M Ω to 2 G Ω. These findings can have important consequences in DNA-based molecular electronics and direct label-free detection of DNA hybridization.
Resistance to downy mildew in pearl millet is associated with increased phenylalanine ammonia lyase activity
Authors: Geetha N. P. , Amruthesh K. N. , Sharathchandra R. G. , Shetty H. S.
Published: April 2005

Phenylalanine ammonia lyase (PAL) activity was studied in pearl millet cultivars with different levels of resistance to the downy mildew disease caused by Sclerospora graminicola, an important oomycete pathogen. PAL activity was elevated in resistant host cultivar and decreased in susceptible cultivars following downy mildew pathogen infection. The enzyme activation varied between cultivars and was correlated with the degree of resistance to downy mildew disease. The induction of PAL as a response to pathogen inoculation was further corroborated by a time-course study in seedlings and cultured cells of pearl millet. The level of PAL activity was highest at 1.5 h in cultured cells and 4 h in seedlings of resistant host cultivar after inoculation with Sclerospora graminicola. Further studies on PAL activity in different tissues of seedlings showed highest enzyme activity in the young growing region of the root of the resistant host cultivars. The accumulation of wall-bound phenolics and lignin was higher in the resistant cultivar seedlings as evidenced by phloroglucinol–HCl staining and p-coumaric acid assay. The temporal changes in lignin concentration and the concentration of soluble phenolics were greater in root tissues of resistant cultivars than in those of susceptible cultivars. Treatment of resistant seedlings with a PAL inhibitor, α-aminooxy-β-phenylpropionic acid, resulted in the enhancement of the enzyme activity, whereas in the presence of 1 mm trans-cinnamic acid the pathogen-induced PAL was completely inhibited. Treatment of pearl millet seedlings with exogenously applied PAL inhibitors induced downy mildew disease susceptibility in the resistant pearl millet cultivar, consistent with direct involvement of PAL in downy mildew resistance. Results are discussed with respect to the presumed importance of host phenolic compounds and lignin accumulation and its relation to PAL activation as a response to the pathogen infection.
Photoperiod during stem elongation in wheat: is its impact on fertile floret and grain number determination similar to that of radiation?
Authors: González Fernanda G. , Slafer Gustavo A. , Miralles Daniel J.
Published: April 2005

Increasing duration of stem elongation by exposure to short photoperiod would result in higher spike dry weight at anthesis, which is positively associated with the number of fertile florets and grains in wheat. However, it is not easy to determine whether photoperiod effects on fertile florets and grains are only mediated by assimilate supply to the growing spike when spike weight variation is attained only with photoperiod treatments. The aim of this study was to determine whether photoperiod effects on number of fertile florets and grains may be direct, that is, not mediated by assimilate supply, by comparing the magnitude of photoperiod effects with those of shading the canopy. Spike dry weight at anthesis was changed through the factorial combination of different photoperiod (natural and 6 h extended photoperiod) and shading (un-shaded and 67 ± 3% shaded) treatments during stem elongation of Buck Manantial, a cultivar known for its photoperiod sensitivity in this phase. Both treatments modified spike dry weight at anthesis and the number of fertile florets and grains, independently. When duration of stem elongation was lengthened by exposure to natural photoperiod and when incident radiation was high, spike dry weight at anthesis increased by 33% (NP+0 v. NP+6) and 27% (un-shaded v. shaded), respectively. The number of fertile florets increased similarly to spike dry weight (34% NP+0 v. NP+6 and 28% un-shaded v. shaded) resulting in higher number of grains. Most photoperiod effects on the number of fertile florets and, consequently, on the number of grains, were mediated by assimilate supply to the growing spike as the same relationship between the number of fertile florets and spike dry weight at anthesis was observed for photoperiod and shading treatments (R2 = 0.99, P<0.05).
The role and the interrelationship of hydrogen peroxide and nitric oxide in the UV-B-induced stomatal closure in broad bean
Authors: He Jun-Min , Xu Hua , She Xiao-Ping , Song Xi-Gui , Zhao Wen-Ming
Published: April 2005

Previous studies have showed that UV-B can stimulate closure as well as opening of stomata. However, the mechanism of this complex effect of UV-B is not clear. The purpose of this paper is to investigate the role and the interrelationship of H2O2 and NO in UV-B-induced stomatal closure in broad bean (Vicia faba L.). By epidermal strip bioassay and laser-scanning confocal microscopy, we observed that UV-B-induced stomatal closure could be largely prevented not only by NO scavenger c-PTIO or NO synthase (NOS) inhibitor l-NAME, but also by ascorbic acid (ASC, an important reducing substrate for H2O2 removal) or catalase (CAT, the H2O2 scavenger), and that UV-B-induced NO and H2O2 production in guard cells preceded UV-B-induced stomatal closure. These results indicate that UV-B radiation induces stomatal closure by promoting NO and H2O2 production. In addition, c-PTIO, l-NAME, ASC and CAT treatments could effectively inhibit not only UV-B-induced NO production, but also UV-B-induced H2O2 production. Exogenous H2O2-induced NO production and stomatal closure were partly abolished by c-PTIO and l-NAME. Similarly, exogenous NO donor sodium nitroprusside-induced H2O2 production and stomatal closure were also partly reversed by ASC and CAT. These results show a causal and interdependent relationship between NO and H2O2 during UV-B-regulated stomatal movement. Furthermore, the l-NAME data also indicate that the NO in guard cells of Vicia faba is probably produced by a NOS-like enzyme.
Identification of a SAR8.2 gene in the susceptible host response of Nicotiana benthamiana to Colletotrichum orbiculare
Authors: Shan Xue Chan , Goodwin Paul H.
Published: April 2005

A SAR8.2 gene, NbSAR8.2m, was obtained from a PCR-selected cDNA subtraction library constructed from mRNA of Nicotiana benthamiana Domin. infected with Colletotrichum orbiculare (Berk & Mont.) von Arx. It is the first SAR8.2 gene described from N. benthamiana and shows relatively high similarity in both the coding and 3′-UTR to NtSAR8.2m of Nicotiana tabacum L. Expression of NbSAR8.2m occurred in healthy plants but was induced 8-fold following infection by C. orbiculare. Virus-induced gene silencing of NbSAR8.2m reduced its expression and resulted in the development of disease symptoms 24 h earlier than in control plants, indicating that NbSAR8.2m affects the length of the biotrophic phase of infection. Both NtSAR8.2m and NbSAR8.2m are unique among the SAR8.2 genes in that they encode for four cysteines near the C-terminus. The conserved cysteines of SAR8.2 genes may indicate roles in stress responses, defence reactions, metal ion homeostasis or other processes.
Parallelism of amino acid changes at the RH1 affecting spectral sensitivity among deep-water cichlids from Lakes Tanganyika and Malawi
Authors: Sugawara Tohru , Terai Yohey , Imai Hiroo , Turner George F. , Koblmüller Stephan , Sturmbauer Christian , Shichida Yoshinori , Okada Norihiro
Published: April 2005

Many examples of the appearance of similar traits in different lineages are known during the evolution of organisms. However, the underlying genetic mechanisms have been elucidated in very few cases. Here, we provide a clear example of evolutionary parallelism, involving changes in the same genetic pathway, providing functional adaptation of RH1 pigments to deep-water habitats during the adaptive radiation of East African cichlid fishes. We determined the RH1 sequences from 233 individual cichlids. The reconstruction of cichlid
RH1
pigments with 11-
cis
-retinal from 28 sequences showed that the absorption spectra of the pigments of nine species were shifted toward blue, tuned by two particular amino acid replacements. These blue-shifted RH1 pigments might have evolved as adaptations to the deep-water photic environment. Phylogenetic evidence indicates that one of the replacements, A292S, has evolved several times independently, inducing similar functional change. The parallel evolution of the same mutation at the same amino acid position suggests that the number of genetic changes underlying the appearance of similar traits in cichlid diversification may be fewer than previously expected.