bluuebunny/crossref_metadata_2025 huggingface dataset:
STUDY OF BLAST DNA MATCHING TOOLKITS
Authors: NG E. Y.-K. , TAY L. L.
Published: August 2004

The beginning of bioinformatics saw the development of algorithms that enabled the storage of nucleic acid and protein sequences in the form of annotated databases in a manner that would allow researchers to exchange information about gene and protein sequences easily and quickly. Databases are growing extremely fast, hence it is essential to use the current databases, which are easily available on the Web.

This tutorial deals with the concept of DNA matching by using BLAST programs such as BLASTN and MEGABLAST to perform similarity sequence search and to evaluate their relative effectiveness. Interpretation of the BLAST results is done. Comparisons between the two algorithms are included based on varying parameters such as word sizes, query sequences length and gap X drop-off values, etc. It is found that as the word size increases, the computation time for both BLASTN and MEGABLAST algorithms decreases. BLASTN is more sensitive than MEGABLAST since it uses a shorter default word size of 11 as compared to MEGABLAST, which uses a default word size of 28. The search strategy offers a tradeoff between speed and sensitivity. As for BLAST 2 Sequences, MEGABLAST could perform better than BLASTN only for large word sizes greater than or equal to 16 and for longer sequences.
INTELLECTUAL CAPITAL AND PERFORMANCE OF NEW VENTURE HIGH-TECH FIRMS
Authors: JUMA NORMA , PAYNE G. TYGE
Published: August 2004

Intellectual capital (IC) has been proposed as an essential factor for organizational survival and maintenance of competitive strength. However, there has been very limited consensus on what encompasses IC and how it can best be conceptualized and measured. Further, very little empirical work has specifically examined the relationship between IC and financial performance. Given these shortcomings, this paper focuses first on the impact IC has on performance and secondly on the role strategic alliances may have on this relationship.

While we argue that IC will impact performance, we anticipate this relationship will be moderated by strategic alliances and other inter-firm collaborations. Findings reveal interesting relationships that suggest further effort should be placed on the conceptualization and measurement of IC, specifically regarding its relationship to firm performance.
THE PROCESS OF NEW SERVICE DEVELOPMENT — ISSUES OF FORMALIZATION AND APPROPRIABILITY
Authors: DOLFSMA WILFRED
Published: August 2004

Services form an important part of the economy today. Innovation for service firms is as important as for manufacturing, but the innovation process for service firms is comparatively little studied. In this paper, I review the literature there is on the innovation process for service firms, and make two suggestions for formalizing that process. The common thought that service firms do not innovate does not hold. Innovation is, however, often ad hoc for services, and it can therefore be difficult to measure firms' innovation efforts. These points are related to issues of appropriability of the benefits of innovation in services. The two issues primarily discussed in this paper — the possibilities of formalizing and appropriating in case of new service development — are central issues for service firms. It is here that this paper offers some contributions to the existing literature; it does not so much present an overview thereof.
Skeletal muscle atrophy leads to loss and dysfunction of muscle precursor cells
Authors: Mitchell Patrick O. , Pavlath Grace K.
Published: August 2004

Atrophy of skeletal muscle leads to decreases in myofiber size and nuclear number; however, the effects of atrophic conditions on muscle precursor cells (MPC) are largely unknown. MPC lie outside myofibers and represent the main source of additional myonuclei necessary for muscle growth and repair. In the present study, we examined the properties of MPC after hindlimb suspension (HS)-induced atrophy and subsequent recovery of the mouse hindlimb muscles. We demonstrated that the number of MPC in atrophied muscles was decreased. RT-PCR analysis of cells isolated from atrophied muscles indicated that several mRNA characteristic of the myogenic program in MPC were absent. Cells isolated from atrophied muscles failed to properly proliferate and undergo differentiation into multinucleated myotubes. Thus atrophy led to a decrease in MPC and caused dysfunction in those MPC that remained. Upon regrowth of the atrophied muscles, these deleterious effects were reversed. Our data suggest that preventing loss or dysfunction of MPC may be a new pharmacological target during muscle atrophy.
Purinergic component of mechanosensory transduction is increased in a rat model of colitis
Authors: Wynn Gregory , Ma Bei , Ruan Huai Zhen , Burnstock Geoffrey
Published: August 2004

ATP contributes to mechanosensory transduction in the rat colorectum. P2X3 receptors are present on dorsal root ganglia (DRG) neurons that supply this area of the gut. Previous studies have shown an increased role for ATP in inflamed tissues. We aimed to investigate whether an increased purinergic component exists during mechanosensory transduction in a rat model of colitis. An in vitro rat colorectal preparation was used to investigate whether distension increased ATP release and to evaluate the role of purinergic antagonists in distension-evoked sensory discharges in the pelvic nerve in normal and colitis preparations. DRG neuron purinoceptors were also studied. Distension-evoked responses in the colitis model were attenuated to a significantly greater extent by 2′,3′- O-trinitrophenyl-ATP and pyridoxyl 5-phosphate 6-azophenyl-2′,4′-disulfonic acid. Inflammation caused augmented distension-evoked sensory nerve excitation after application of ATP and α,β-methylene ATP. Single-fiber analysis confirmed that mean firing per unit was increased. Distension-evoked increases in ATP release from epithelial cells were substantially higher. The number of DRG neurons responding to ATP and the number of those staining for the P2X3 receptor, particularly those containing calcitonin gene-related peptide, were increased. Adenosine, after ectoenzymatic breakdown of ATP, is involved to a lesser degree in the longer-lasting distension-evoked sensory discharge, suggesting reduced ATPase activity. It was therefore concluded that ATP has an enhanced role in mechanosensory transduction in the inflamed rat colorectum. The underlying mechanisms appear to involve increased distension-evoked release of ATP as well as an increase in the number of DRG neurons supplying the colorectum expressing P2X3 receptors, especially those containing calcitonin gene-related peptide.
Mechanism of internal anal sphincter relaxation by CORM-1, authentic CO, and NANC nerve stimulation
Authors: Rattan Satish , Haj Rany Al , De Godoy Márcio A. F.
Published: August 2004

The present studies compared the effects of CO-releasing molecule (CORM-1), authentic CO, and nonadrenergic noncholinergic (NANC) nerve stimulation in the internal anal sphincter (IAS). Functional in vitro experiments and Western blot studies were conducted in rat IAS smooth muscle. We examined the effects of CORM-1 (50–600 μM) and authentic CO (5–100 μM) and NANC nerve stimulation by electrical field stimulation (EFS; 0.5–20 Hz, 0.5-ms pulse, 12 V, 4-s train). The experiments were repeated after preincubation of the tissues with the neurotoxin TTX, the guanylate cyclase inhibitor 1 H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ), the selective heme oxygenase (HO) inhibitor tin protoporphyrin IX (SnPP-IX), the nitric oxide synthase inhibitor Nω-nitro-l-arginine (l-NNA), and SnPP-IX + l-NNA. We also investigated the effects of the HO substrate hematin (100 μM). CORM-1, as well as CO, produced concentration-dependent IAS relaxation, whereas hematin had no effect. TTX abolished and l-NNA significantly blocked IAS relaxation by EFS without any effect on CORM-1 and CO. ODQ blocked IAS relaxation by CORM-1, authentic CO, and EFS. SnPP-IX had no significant effect on IAS relaxation by CORM-1, CO, or EFS. The presence of neuronal nitric oxide synthase, HO-1, and HO-2 in IAS smooth muscle was confirmed by Western blot studies. CORM-1 and CO, as well as NANC nerve stimulation, produced IAS relaxation via guanylate cyclase/cGMP-dependent protein kinase activation. The advent of CORM-1 with potent effects in the IAS has significant implications in anorectal motility disorders with regard to pathophysiology and therapeutic potentials.
Role of small GTP binding proteins in the growth-promoting and antiapoptotic actions of gastrin
Authors: Stepan Vinzenz , Ramamoorthy Saravanan , Pausawasdi Nonthalee , Logsdon Craig D. , Askari Frederick K. , Todisco Andrea
Published: August 2004

G17 has growth promoting and antiapoptotic effects on the AR4–2J pancreatic acinar cell line. We previously reported that whereas MAPK regulates G17-stimulation of AR4–2J cell proliferation, Akt mediates the antiapoptotic action of G17. We examined the signal-transduction pathways mediating G17 stimulation of AR4–2J cell growth and survival. G17 activated the small GTP binding proteins Ras, Rac, Rho, and Cdc42. Transduction of the cells with adenoviral vectors expressing dominant negative Akt, Ras, Rho, and Cdc42 but not dominant negative Rac inhibited AR4–2J cell proliferation and survival. Both exoenzyme C3 from Clostridium botulinum (C3), a toxin known to inactivate Rho, and PD98059, a MAPK inhibitor, reversed G17 inhibition of AR4–2J cell apoptosis. G17 induction of Akt activation was reduced by >60% by both dominant negative Ras and Rho and by 30% by dominant negative Cdc42. In contrast, G17-stimulated MAPK activation was blocked by >80% by dominant negative Ras but not by dominant negative Rho and Cdc42. Similar results were observed in the presence of C3. Dominant negative Rac failed to affect G17 induction of both Akt and MAPK, whereas it inhibited sorbitol by almost 50% but not G17-stimulated activation of p38 kinase. Thus G17 promotes AR4–2J cell growth and survival through the activation of multiple GTP binding proteins, which, in turn, regulate different protein kinase cascades. Whereas Ras activates Akt and MAPK, Rho and Cdc42 appear to regulate Akt and possibly other as yet unidentified kinases mediating the growth-stimulatory actions of G17.
Mechanisms of polymicrobial sepsis-induced ileus
Authors: Overhaus Marcus , Tögel Sandra , Pezzone Michael A. , Bauer Anthony J.
Published: August 2004

Sepsis frequently occurs after hemorrhage, trauma, burn, or abdominal surgery and is a leading cause of morbidity and mortality in severely ill patients. We performed experiments to delineate intestinal molecular and functional motility consequences of polymicrobial sepsis in the clinically relevant cecal ligation and puncture (CLP) sepsis model. CLP was performed on male Sprague-Dawley rats. Gastrointestinal transit, colonic in vivo pressure recordings, and in vitro muscle contractions were recorded. Histochemistry was performed for macrophages, monocytes, and neutrophils. Inflammatory gene expressions were quantified by real-time RT-PCR. CLP delayed gastrointestinal transit, decreased colonic pressures, and suppressed in vivo circular muscle contractility of the jejunum and colon over a 4-day period. A leukocytic infiltrate of monocytes and neutrophils developed over 24 h. Real-time RT-PCR demonstrated a significant temporal elevation in IL-6, IL-1β, monocyte chemoattractant protein-1, and inducible nitric oxide synthase, with higher expression levels of IL-6 and inducible nitric oxide synthase in colonic extracts compared with small intestine. Polymicrobial CLP sepsis induces a complex inflammatory response within the intestinal muscularis with the recruitment of leukocytes and elaboration of mediators that inhibit intestinal muscle function. Differences were elucidated between endotoxin and CLP models of sepsis, as well as a heterogeneous regional response of the gastrointestinal tract to CLP. Thus the intestine is not only a source of bacteremia but also an important target of bacterial products with major functional consequences to intestinal motility and the generation of cytokines, which participate in the development of multiple organ failure.
Genetic manipulation of calcium-handling proteins in cardiac myocytes. I. Experimental studies
Authors: Coutu Pierre , Metzger Joseph M.
Published: August 2004

Two genetic experimental approaches, de novo expression of parvalbumin (Parv) and overexpression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a), have been shown to increase relaxation rates in myocardial tissue. However, the relative effect of Parv and SERCA2a on systolic function and on β-adrenergic responsiveness at varied pacing rates is unknown. We used gene transfer in isolated rat adult cardiac myocytes to gain a fuller understanding of Parv/SERCA2a function. As demonstrated previously, when Parv is expressed in elevated concentration (>0.1 mM), the transduced myocytes showed a reduction in sarcomere-shortening amplitude: 129 ± 17, 81 ± 8, and 149 ± 14 nm for control, Parv, and SERCA2a, respectively. At physiological temperature, shortening amplitude responses of Parv and SERCA2a myocytes to the β-adrenergic agonist isoproterenol (Iso) were not statistically different from that of control myocytes. However, in SERCA2a myocytes, in which baseline was slightly elevated and the Iso-stimulated value was slightly lower, the increase in shortening was slightly less than in Parv or control myocytes: 108 ± 14, 169 ± 39, and 34 ± 12% for control, Parv, and SERCA2a, respectively. In another test set, Parv myocytes had the strongest early postrest potentiation among all groups studied (rest time = 2–10 s), and SERCA2a myocytes were the least sensitive to variations in stimulation rhythm. To replicate the deficient Ca2+ removal observed in heart failure, we used 150 nM thapsigargin. Under these conditions, control myocytes exhibited slowed relaxation, whereas Parv myocytes retained their rapid kinetics, showing that Parv is still able to control relaxation, even when SERCA2a function is impaired.
Acetylcholine and bradykinin trigger preconditioning in the heart through a pathway that includes Akt and NOS
Authors: Krieg Thomas , Qin Qining , Philipp Sebastian , Alexeyev Mikhail F. , Cohen Michael V. , Downey James M.
Published: August 2004

In the rabbit heart, bradykinin and ACh trigger preconditioning by a mechanism involving ATP-sensitive potassium channel-dependent production of reactive oxygen species (ROS). Recent evidence indicates that the pathway by which bradykinin causes ROS generation includes nitric oxide synthase (NOS) and protein kinase G (PKG). On the other hand, Akt was shown to be essential for ACh to generate ROS. This study determines whether these two G-coupled receptor agonists indeed have similar signaling targets, i.e., whether Akt is involved in bradykinin's pathway and whether NOS is involved in ACh's pathway. Isolated adult rabbit cardiomyocytes were incubated for 15 min in reduced MitoTracker red, which becomes fluorescent only after exposure to ROS. Bradykinin (400 nM) and ACh (250 μM) caused a 51.4 ± 14.8% and 39.8 ± 11.7% increase, respectively, in ROS production ( P < 0.005). Coincubation of either agonist with Akt inhibitor (20 μM) or infection of cells with an adenovirus containing dominant negative Akt abolished this increase. The NO donor S-nitroso- N-acetyl penicillamine (SNAP, 1 μM) also increased the ROS signal by 40.8 ± 15.7%, but this increase was unaffected by Akt inhibitor (39.0 ± 6.4%), implying that Akt is upstream of NOS. ACh-induced ROS production could be abolished by either of the NOS inhibitors Nω-monomethyl-l-arginine monoacetate (100 μM) and l- N5-(1-iminoethyl)ornithine hydrochloride (l-NIO, 5 μM). l-NIO also blocked the anti-infarct effect of ACh (550 μM) in isolated rabbit hearts exposed to 30 min of regional ischemia. We conclude that both bradykinin and ACh trigger ROS generation by sequentially activating Akt and NOS.
Enhanced protein synthetic capacity in Atlantic cod (Gadus morhua) is associated with temperature-induced compensatory growth
Authors: Treberg Jason R. , Hall Jennifer R. , Driedzic William R.
Published: August 2004

Atlantic cod ( Gadus morhua) were held either at seasonal ambient temperatures (−0.3 to 11°C) or at a relatively constant control temperature (8–11°C) to investigate aspects of protein synthesis during a period of compensatory growth. Protein synthesis rate, total RNA, and RNA-specific protein synthesis rate were determined in white muscle and liver when ambient temperatures were −0.3, 4.5, and 11°C in February, June, and July, respectively. To allow for comparisons between treatment temperatures, fish were also acutely transferred to a comparable assay temperature in February and June. Over the transition from 4.5 to 11°C (June to July), the ambient-held cod had a significant increase in size and a substantially higher growth rate relative to control-held fish over the same period, consistent with cold-induced compensatory growth. During the onset of this enhanced growth, in June when ambient temperature was ∼4.5°C, ambient-held fish elevated their capacity for protein synthesis in the white muscle and liver via elevation of the RNA content. When ambient temperature reached the same point as for the control fish (11°C), the rate of white muscle protein synthesis remained higher in the ambient-held vs. that in the control-held fish, a process facilitated by elevated RNA content and greater RNA-specific rate of protein synthesis. In the liver, all measured characteristics of protein synthesis were the same for ambient and control fish in July. The latter suggests that compensatory growth may be in part explained by improved efficiency of protein synthesis.
Some theorems on the explicit evaluation of Ramanujan′s theta‐functions
Authors: Baruah Nayandeep Deka , Bhattacharyya P.
Published: August 2004

Bruce C. Berndt et al. and Soon‐Yi Kang have proved many of Ramanujan′s formulas for the explicit evaluation of the Rogers‐Ramanujan continued fraction and theta‐functions in terms of Weber‐Ramanujan class invariants. In this note, we give alternative proofs of some of these identities of theta‐functions recorded by Ramanujan in his notebooks and deduce some formulas for the explicit evaluation of his theta‐functions in terms of Weber‐Ramanujan class invariants.
A remark on the extension of the concept of incidence algebras to nonlocally finite partially ordered sets
Authors: Eke Boniface I.
Published: August 2004

An incidence algebra of a nonlocally finite partially ordered set Q is a very rare concept, perhaps nonexistent. In this note, we will attempt to construct such an algebra.
The axisymmetric Boussinesq‐type problem for a half‐space under optimal heating of arbitrary profile
Authors: Rokne J. , Singh B. M. , Dhaliwal R. S. , Vrbik J.
Published: August 2004

A solution of the axisymmetric Boussinesq‐type problem is derived for transient thermal stresses in a half‐space under heating by using the Laplace and Hankel transforms. An analytical method is developed to predict the temperature field that satisfies the prescribed mechanical conditions. Several simple shapes of punches of arbitrary profile are considered and an expression for the total load is derived to achieve penetration. The numerical results for the temperature and the total load on the punch are shown graphically.
On bounded cohomology of amalgamated products of groups
Authors: Erovenko Igor V.
Published: August 2004

We investigate the structure of the singular part of the second bounded cohomology group of amalgamated products of groups by constructing an analog of the initial segment of the Mayer‐Vietoris exact cohomology sequence for the spaces of pseudocharacters.
Effective Memetic Algorithms for VLSI Design = Genetic Algorithms + Local Search + Multi-Level Clustering
Authors: Areibi Shawki , Yang Zhen
Published: August 2004

Combining global and local search is a strategy used by many successful hybrid optimization approaches. Memetic Algorithms (MAs) are Evolutionary Algorithms (EAs) that apply some sort of local search to further improve the fitness of individuals in the population. Memetic Algorithms have been shown to be very effective in solving many hard combinatorial optimization problems. This paper provides a forum for identifying and exploring the key issues that affect the design and application of Memetic Algorithms. The approach combines a hierarchical design technique, Genetic Algorithms, constructive techniques and advanced local search to solve VLSI circuit layout in the form of circuit partitioning and placement. Results obtained indicate that Memetic Algorithms based on local search, clustering and good initial solutions improve solution quality on average by 35% for the VLSI circuit partitioning problem and 54% for the VLSI standard cell placement problem.
Developmental modulation ofFab-7boundary function
Authors: Schweinsberg Susan E. , Schedl Paul
Published: August 2004

The Fab-7 boundary functions to ensure the autonomous activity of the iab-6 and iab-7 cis-regulatory domains in the Drosophila Bithorax Complex from early embryogenesis through to the adult stage. Although Fab-7 is required only for the proper development of a single posterior parasegment, it is active in all tissues and stages of development that have been examined. In this respect, Fab-7resembles conventional constitutive boundaries in flies and other eukaryotes that act through ubiquitous cis-elements and trans-acting factors. Surprisingly, however, we find that the constitutive activity of Fab-7 is generated by combining sub-elements with developmentally restricted boundary function. We provide in vivo evidence that the Fab-7 boundary contains separable regions that function at different stages of development. These findings suggest that the units (domains) of genetic regulation that boundaries delimit can expand or contract by switching insulator function off or on in a temporally regulated fashion.
Cell interactions and planar polarity in the abdominal epidermis ofDrosophila
Authors: Lawrence Peter A. , Casal José , Struhl Gary
Published: August 2004

The integument of the Drosophila adult abdomen bears oriented hairs and bristles that indicate the planar polarity of the epidermal cells. We study four polarity genes, frizzled (fz), prickle (pk), Van gogh/strabismus(Vang/stbm) and starry night/flamingo (stan/fmi),and note what happens when these genes are either removed or overexpressed in clones of cells. The edges of the clones are interfaces between cells that carry different amounts of gene products, interfaces that can cause reversals of planar polarity in the clone and wild-type cells outside them. To explain,we present a model that builds on our earlier picture of a gradient of X, the vector of which specifies planar polarity and depends on two cadherin proteins, Dachsous and Fat. We conjecture that the X gradient is read out,cell by cell, as a scalar value of Fz activity, and that Pk acts in this process, possibly to determine the sign of the Fz activity gradient.

We discuss evidence that cells can compare their scalar readout of the level of X with that of their neighbours and can set their own readout towards an average of those. This averaging, when it occurs near the edges of clones,changes the scalar response of cells inside and outside the clones, leading to new vectors that change polarity. The results argue that Stan must be present in both cells being compared and acts as a conduit between them for the transfer of information. And also that Vang assists in the receipt of this information. The comparison between neighbours is crucial, because it gives the vector that orients hairs – these point towards the neighbour cell that has the lowest level of Fz activity.

Recently, it has been shown that, for a limited period shortly before hair outgrowth in the wing, the four proteins we study, as well as others, become asymmetrically localised in the cell membrane, and this process is thought to be instrumental in the acquisition of cell polarity. However, some results do not fit with this view – we suggest that these localisations may be more a consequence than a cause of planar polarity.
The splanchnic mesodermal plate directs spleen and pancreatic laterality,and is regulated byBapx1/Nkx3.2
Authors: Hecksher-Sørensen Jacob , Watson Robert P. , Lettice Laura A. , Serup Palle , Eley Lorraine , De Angelis Carlo , Ahlgren Ulf , Hill Robert E.
Published: August 2004

The mechanism by which left-right (LR) information is interpreted by organ primordia during asymmetric morphogenesis is largely unknown. We show that spleen and pancreatic laterality is dependent on a specialised, columnar mesodermal-derived cell layer referred to here as the splanchnic mesodermal plate (SMP). At early embryonic stages, the SMP is bilateral, surrounding the midline-located stomach and dorsal pancreatic bud. Under control of the LR asymmetry pathway, the left SMP is maintained and grows laterally. Mice carrying the dominant hemimelia (Dh) mutation lack the SMP. Significantly, the mice are asplenic and the pancreas remains positioned along the embryonic midline. In the absence of Fgf10 expression, the spleno-pancreatic mesenchyme and surrounding SMP grow laterally but contain no endodermal component, showing that leftward growth is autonomous and independent of endoderm. In the Bapx1–/–mutants, the SMP is defective. Normally, the SMP is a source for both Fgf9 and Fgf10; however, in the Bapx1 mutant, Fgf10 expression is downregulated and the dorsal pancreas remains at the midline. We conclude that the SMP is an organiser responsible for the leftward growth of the spleno-pancreatic region and that Bapx1 regulates SMP functions required for pancreatic laterality.
Dynactin is required to maintain nuclear position within postmitoticDrosophilaphotoreceptor neurons
Authors: Whited Jessica L. , Cassell Andre , Brouillette Monique , Garrity Paul A.
Published: August 2004

How a nucleus is positioned within a highly polarized postmitotic animal cell is not well understood. In this work, we demonstrate that the Dynactin complex (a regulator of the microtubule motor protein Dynein) is required to maintain the position of the nucleus within post-mitotic Drosophila melanogaster photoreceptor neurons. We show that multiple independent disruptions of Dynactin function cause a relocation of the photoreceptor nucleus toward the brain, and that inhibiting Dynactin causes the photoreceptor to acquire a bipolar appearance with long leading and trailing processes. We find that while the minus-end directed motor Dynein cooperates with Dynactin in positioning the photoreceptor nucleus, the plus-end directed microtubule motor Kinesin acts antagonistically to Dynactin. These data suggest that the maintenance of photoreceptor nuclear position depends on a balance of plus-end and minus-end directed microtubule motor function.
The involvement of Frodo in TCF-dependent signaling and neural tissue development
Authors: Hikasa Hiroki , Sokol Sergei Y.
Published: August 2004

Frodo is a novel conserved regulator of Wnt signaling that has been identified by its association with Dishevelled, an intracellular component of Wnt signal transduction. To understand further how Frodo functions, we have analyzed its role in neural development using specific morpholino antisense oligonucleotides. We show that Frodo and the closely related Dapper synergistically regulate head development and morphogenesis. Both genes were cell-autonomously required for neural tissue formation, as defined by the pan-neural markers sox2 and nrp1. By contrast,β-catenin was not required for pan-neural marker expression, but was involved in the control of the anteroposterior patterning. In the mesoderm,Frodo and Dapper were essential for the expression of the organizer genes chordin, cerberus and Xnr3, but they were not necessary for the expression of siamois and goosecoid,established targets of β-catenin signaling. Embryos depleted of either gene showed a decreased transcriptional response to TCF3-VP16, aβ-catenin-independent transcriptional activator. Whereas the C terminus of Frodo binds Dishevelled, we demonstrate that the conserved N-terminal domain associates with TCF3. Based on these observations, we propose that Frodo and Dapper link Dsh and TCF to regulate Wnt target genes in a pathway parallel to that of β-catenin.
The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor
Authors: von Rotz Ruth C. , Kohli Bernhard M. , Bosset Jérôme , Meier Michelle , Suzuki Toshiharu , Nitsch Roger M. , Konietzko Uwe
Published: August 2004

The physiological functions of the beta-amyloid precursor protein (APP) may include nuclear signaling. To characterize the role of the APP adaptor proteins Fe65, Jip1b, X11α (MINT1) and the chromatin-associated protein Tip60, we analyzed their interactions by confocal microscopy and co-immunoprecipitations. AICD corresponding to S3-cleaved APP bound to Fe65 that transported it to nuclei and docked it to Tip60. These proteins formed AICD-Fe65-Tip60 (AFT) complexes that were concentrated in spherical nuclear spots. γ-Secretase inhibitors prevented AFT-complex formation with AICD derived from full-length APP. The APP adaptor protein Jip1b also transported AICD to nuclei and docked it to Tip60, but AICD-Jip1b-Tip60 (AJT) complexes had different, speckle-like morphology. By contrast, X11α trapped AICD in the cytosol. Induced AICD expression identified the APP-effector genes APP, BACE, Tip60, GSK3β and KAI1, but not the Notch-effector gene Hes1 as transcriptional targets. These data establish a role for APP in nuclear signaling, and they suggest that therapeutic strategies designed to modulate the cleavage of APP affect AICD-dependent signaling.
Zinc metalloproteinase-mediated cleavage of the human Nogo-66 receptor
Authors: Walmsley Adrian R. , McCombie Gregor , Neumann Ulf , Marcellin David , Hillenbrand Rainer , Mir Anis K. , Frentzel Stefan
Published: August 2004

The central nervous system myelin components oligodendrocyte-myelin glycoprotein, myelin-associated glycoprotein and the Nogo-66 domain of Nogo-A inhibit neurite outgrowth by binding the neuronal glycosyl-phosphatidylinositol-anchored Nogo-66 receptor (NgR) that transduces the inhibitory signal to the cell interior via a transmembrane co-receptor, p75NTR. Here, we demonstrate that human NgR expressed in human neuroblastoma cells is constitutively cleaved in a post-ER compartment to generate a lipid-raft associated C-terminal fragment that is present on the cell surface and a soluble N-terminal fragment that is released into the medium. Mass spectrometric analysis demonstrated that the N-terminal fragment terminated just after the C-terminus of the ligand-binding domain of NgR. In common with other shedding mechanisms, the release of this fragment was blocked by a hydroxamate-based inhibitor of zinc metalloproteinases, but not by inhibitors of other protease classes and up-regulated by treatment with the cellular cholesterol depleting agent methyl-β-cyclodextrin. The N-terminal fragment bound Nogo-66 and blocked Nogo-66 binding to cell surface NgR but failed to associate with p75NTR, indicative of a role as a Nogo-66 antagonist. Furthermore, the N- and C-terminal fragments of NgR were detectable in human brain cortex and the N-terminal fragment was also present in human cerebrospinal fluid, demonstrating that NgR proteolysis occurs within the human nervous system. Our findings thus identify a potential cellular mechanism for the regulation of NgR function at the level of the receptor.
Breakpoint cluster regions of the AML-1 and ETO genes contain MAR elements and are preferentially associated with the nuclear matrix in proliferating HEL cells
Authors: Iarovaia Olga V. , Shkumatov Petr , Razin Sergey V.
Published: August 2004

The spatial organization in interphase nuclei of the breakpoint cluster regions (BCRs) of the AML-1 and ETO genes frequently participating in reciprocal t(8;21) translocations was studied using cytological and biochemical approaches. Both BCRs were found to be localized preferentially, but not exclusively, to the nuclear matrix, as shown by hybridization of specific probes with nuclear halos. This association was not related to transcription, because the transcribed regions of both genes located far from BCRs were located preferentially in loop DNA, as shown by in situ hybridization. The sites of association with the nuclear matrix of the intensely transcribed AML-1 gene were mapped also using the biochemical PCR-based approach. Only the BCR was found to be associated with the nuclear matrix, whereas the other transcribed regions of this gene turned out to be positioned randomly in respect to the nuclear matrix. The data are discussed in the framework of the hypothesis postulating that the nuclear matrix plays an important role in determining the positions of recombination-prone areas.
Dominant negative effect of connexin33 on gap junctional communication is mediated by connexin43 sequestration
Authors: Fiorini Céline , Mograbi Baharia , Cronier Laurent , Bourget Isabelle , Decrouy Xavier , Nebout Marielle , Ferrua Bernard , Malassine André , Samson Michel , Fénichel Patrick , Segretain Dominique , Pointis Georges
Published: August 2004

Gap junctional intercellular communication is involved in the control of cell proliferation and differentiation. Connexin33, a member of the multi-gene family of gap junction proteins, exerts an inhibitory effect on intercellular communication when injected into Xenopus oocytes. However, the molecular mechanisms involved remain to be elucidated. Our results show that connexin33 was only expressed within the seminiferous tubules in the testis. In contrast to the majority of connexins, connexin33 was unphosphorylated. Immunoprecipitation experiments revealed that connexin33 physically interacted with connexin43, mainly with the phosphorylated P1 isoform of connexin43 but not with connexin26 and connexin32, two other connexins expressed in the tubular compartment. In Sertoli cells and COS-7 cells, connexin43 was located at the plasma membrane, whereas in connexin33 transfected cells, the specific association of connexin33/43 was sequestered in the intracellular compartment. High-resolution fluorescent deconvolution microscopy indicated that the connexin33/43 complex was mainly found within early endosomes. Sequestration of connexin33/43 complex was associated with a complete inhibition of the gap junctional coupling between adjacent cells. These findings provide the first evidence of a new mechanistic model by which a native connexin, exerting a dominant negative effect, can inhibit gap junctional intercellular communication. In the testis, connexin33 could exert a specific role on germ cell proliferation by suppressing the regulatory effect of connexin43.
HERC5, a HECT E3 ubiquitin ligase tightly regulated in LPS activated endothelial cells
Authors: Kroismayr Renate , Baranyi Ulrike , Stehlik Christian , Dorfleutner Andrea , Binder Bernd R. , Lipp Joachim
Published: August 2004

By differential screening we isolated genes upregulated in inflammatory cytokine-stimulated human skin microvascular endothelial cells. One of these cDNAs encoded RCC1 (regulator of chromosome condensation 1)-like repeats and a HECT (homologous to E6-AP C-terminus) domain, representing a member of the HERC (HECT and RCC1 domain protein) family of ubiquitin ligases. The mRNA level of this member, HERC5, is specifically upregulated in endothelial cells by the pro-inflammatory cytokines tumor necrosis factor α and interleukin 1β, and by lipopolysaccharide (LPS), but is hardly expressed in other cells of the vascular wall such as primary smooth muscle cells and fibroblasts. Regulation of HERC5 gene expression suggests a critical role for the transcription factor NF-κB. In contrast to mRNA expression HERC5 protein is subject of enhanced degradation upon LPS stimulation of endothelial cells. The time course of LPS-induced changes in HERC5 protein and mRNA levels suggests that the initial drop in HERC5 protein is balanced by increased protein synthesis due to upregulation of HERC5 mRNA. This leads to recovery of HERC5 protein levels within 12 hours of LPS stimulation and points at a tight control of HERC5 protein. To analyze functional activity of this putative member of the ubiquitin-conjugating pathway we performed in vitro assays with different ubiquitin-conjugating enzymes. We found that HERC5 possesses ubiquitin ligase activity and requires the presence of the ubiquitin-conjugating enzyme UbcH5a for its activity. These data show for the first time that a functionally active HECT ubiquitin ligase exhibits a tightly controlled cytosolic level under inflammatory conditions in endothelial cells.
Modulation of intercellular communication in macrophages: possible interactions between GAP junctions and P2 receptors
Authors: Fortes Fabio S. A. , Pecora Iracy L. , Persechini Pedro M. , Hurtado Sandra , Costa Vandir , Coutinho-Silva Robson , Braga Mariane B. Melo , Silva-Filho Fernando C. , Bisaggio Rodrigo C. , de Farias Fernando Pires , Scemes Eliana , de Carvalho Antônio C. Campos , Goldenberg Regina C. S.
Published: August 2004

Gap junctions are connexin-formed channels that play an important role in intercellular communication in most cell types. In the immune system, specifically in macrophages, the expression of connexins and the establishment of functional gap junctions are still controversial issues. Macrophages express P2X7 receptors that, once activated by the binding of extracellular ATP, lead to the opening of transmembrane pores permeable to molecules of up to 900 Da. There is evidence suggesting an interplay between gap junctions and P2 receptors in different cell systems. Thus, we used ATP-sensitive and -insensitive J774.G8 macrophage cell lines to investigate this interplay. To study junctional communication in J774-macrophage-like cells, we assessed cell-to-cell communication by microinjecting Lucifer Yellow. Confluent cultures of ATP-sensitive J774 cells (ATP-s cells) are coupled, whereas ATP-insensitive J774 cells (ATP-i cells), derived by overexposing J774 cells to extracellular ATP until they do not display the phenomenon of ATP-induced permeabilization, are essentially uncoupled. Western-blot and reverse-transcription polymerase chain reaction assays revealed that ATP-s and ATP-i cells express connexin43 (Cx43), whereas only ATP-s cells express the P2X7 receptor. Accordingly, ATP-i cells did not display any detectable ATP-induced current under whole-cell patch-clamp recordings. Using immunofluorescence microscopy, Cx43 reactivity was found at the cell surface and in regions of cell-cell contact of ATP-s cells, whereas, in ATP-i cells, Cx43 immunoreactivity was only present in cytosolic compartments. Using confocal microscopy, it is shown here that, in ATP-s cells as well as in peritoneal macrophages, Cx43 and P2X7 receptors are co-localized to the membrane of ATP-s cells and peritoneal macrophages.
Gyp5p and Gyl1p are involved in the control of polarized exocytosis in budding yeast
Authors: Chesneau Laurent , Dupré Sophie , Burdina Anna , Roger Jérôme , Le Panse Sophie , Jacquet Michel , Cuif Marie-Hélène
Published: August 2004

We report here elements for functional characterization of two members of the Saccharomyces cerevisiae Ypt/Rab GTPase activating proteins family (GAP): Gyp5p, a potent GAP in vitro for Ypt1p and Sec4p, and the protein Ymr192wp/APP2 that we propose to rename Gyl1p (GYp like protein). Immunofluorescence experiments showed that Gyp5p and Gyl1p partly colocalize at the bud emergence site, at the bud tip and at the bud neck during cytokinesis. Subcellular fractionation and co-immunoprecipitation experiments showed that Gyp5p and Gyl1p co-fractionate with post-Golgi vesicles and plasma membrane, and belong to the same protein complexes in both localizations. We found by co-immunoprecipitation experiments that a fraction of Gyp5p interacts with Sec4p, a small GTPase involved in exocytosis, and that a fraction of Gyl1p associates at the plasma membrane with the Gyp5p/Sec4p complexes. We showed also that GYP5 genetically interacts with SEC2, which encodes the Sec4p exchange factor. Examination of the gyp5Δgyl1Δ mutants grown at 13°C revealed a slight growth defect, a secretion defect and an accumulation of secretory vesicles in the small-budded cells. These data suggest that Gyp5p and Gyl1p are involved in control of polarized exocytosis.
The nuclear envelope lamina network has elasticity and a compressibility limit suggestive of a molecular shock absorber
Authors: Dahl Kris Noel , Kahn Samuel M. , Wilson Katherine L. , Discher Dennis E.
Published: August 2004

Mechanical properties of the nuclear envelope have implications for cell and nuclear architecture as well as gene regulation. Using isolated Xenopus oocyte nuclei, we have established swelling conditions that separate the intact nuclear envelope (membranes, pore complexes and underlying lamin filament network) from nucleoplasm and the majority of chromatin. Swelling proves reversible with addition of high molecular mass dextrans. Micropipette aspiration of swollen and unswollen nuclear envelopes is also reversible and yields a network elastic modulus, unaffected by nucleoplasm, that averages 25 mN/m. Compared to plasma membranes of cells, the nuclear envelope is much stiffer and more resilient. Our results suggest that the nuclear lamina forms a compressed network shell of interconnected rods that is extensible but limited in compressibility from the native state, thus acting as a `molecular shock absorber'. In light of the conservation of B-type lamins in metazoan evolution, the mechanical properties determined in this investigation suggest physical mechanisms by which mutated lamins can either destabilize nuclear architecture or influence nuclear responses to mechanical signals in Emery-Dreifuss muscular dystrophy, cardiomyopathy, progeria syndromes (premature `aging') and other laminopathies.
Epigenetic aspects of differentiation
Authors: Arney Katharine L. , Fisher Amanda G.
Published: August 2004

A major challenge in biology is to understand how genetic information is interpreted to direct the formation of specialized tissues within a multicellular organism. During differentiation, changes in chromatin structure and nuclear organization establish heritable patterns of gene expression in response to signals. Epigenetic states can be broadly divided into three categories: euchromatin, constitutive heterochromatin and facultative hetereochromatin. Although the static epigenetic profiles of expressed and silent loci are relatively well characterized, less is known about the transition between active and repressed states. Furthermore, it is important to expand on localized models of chromatin structure at specific genetic addresses to examine the entire nucleus. Changes in nuclear organization, replication timing and global chromatin modifications should be integrated when attempting to describe the epigenetic signature of a given cell type. It is also crucial to examine the temporal aspect of these changes. In this context, the capacity for cellular differentiation reflects both the repertoire of available transcription factors and the accessibility of cis-regulatory elements, which is governed by chromatin structure. Understanding this interplay between epigenetics and transcription will help us to understand differentiation pathways and, ultimately, to manipulate or reverse them.
Thrombophiliediagnostik bei Empfängern von Organtransplantaten / Thrombophilia diagnostics in recipients of organ transplants
Authors: Orth M. , Müller M. , Petros S. , Thiery J.
Published: August 2004

Patients with the highest risk of recurrent thromboses due to inherited or acquired plasmatic factors can be identified by laboratory testing, the so-called "thrombophilia diagnostics". This review summarizes the advantages of thrombophilia diagnostics in organ transplant recipients for the primary prevention of vessel occlusion and transplant dysfunction. On the basis of published studies, the primary prevention of the transplant dysfunction of the kidney is a useful indication for thrombophilia diagnostics while this testing is only of limited use in heart transplant recipients. In patients with a proven hypercoagulable state, appropiate immunosuppressive agents should be chosen with respect to prothrombotic effects and prophylactic anticoagulation should be considered.
Maternal thrombophilia and obstetric complications / Mütterliche Thrombophilie und geburtshilfliche Komplikationen
Authors: Lindhoff-Last E.
Published: August 2004

Women with thrombophilic defects have been shown to
be at increased risk, not only of pregnancy associated
thromboembolism but also of other vascular complications
of pregnancy, including preeclampsia and fetal loss.
First trimester fetal loss is associated with factor V Leiden
mutation, activated protein C resistance without factor V
Leiden mutation and prothrombin G20210A mutation.
Late nonrecurrent fetal loss is associated with factor V
Leiden mutation, prothrombin mutation and protein S
deficiency. Concerning acquired thrombophilia, recurrent
fetal loss is a well-documented finding in patients with
antiphospholipid antibodies. Associations between
thrombophilia polymorphisms and an increased risk of
intrauterine growth restriction have been discussed in
small series of cases but could not be confirmed in large
scale studies. Frequencies for anticardiolipin antibodies
or lupus anticoagulants and antinuclear antibodies were
significantly higher in women with infants small for gestational
age compared to controls. Concerning preeclampsia,
gestational hypertension and thrombophilia, a
number of studies have examined these relationships
with conflicting results. For factor V Leiden, MTHFR
C677T and prothrombin mutation, no association with
preeclampsia was observed, when severe cases were
excluded. If studies were restricted to those of severe
preeclampsia, an association with the factor V Leiden
mutation was apparent and, to a lesser extent, with the
MTHFR-mutation.
For antithrombotic therapy, it was shown that in women
with antiphospholipid syndrome and recurrent pregnancy
loss, unfractionated heparin plus lowdose aspirin
results in significantly better gestational outcome than
lowdose aspirin alone. Concerning therapy of women
with inherited thrombophilia and pregnancy loss, only small, uncontrolled studies are available, demonstrating
improved pregnancy outcome when low molecular
weight heparin (LMWH) is used for treatment.
In conclusion, heritable thrombophilia and the antiphospholipid-syndrome are major causes of fetal loss
after exclusion of other underlying pathologies like chromosomal abnormalities, and screening should be recommended. LMWH with or without aspirin may be used for treatment. There is little value in antenatal screening for prothrombotic polymorphisms to predict the development of small for gestational age infants, preeclampsia or gestational hypertension.