THERAPEUTIC STRATEGIES FOR THE INHIBITION OF INDUCIBLE NITRIC OXIDE SYNTHASE—POTENTIAL FOR A NOVEL CLASS OF ANTI-INFLAMMATORY AGENTS

In recent years, NO, a gas previously considered a potentially toxic chemical, has become established as a diffusible universal messenger mediating cell—cell communication throughout the body. In mammals, NO is a recognized mediator of blood vessel relaxation that helps to maintain blood pressure. In the central nervous system NO acts as a non-conventional neurotransmitter and participates in the establishment of long-term plasticity required for memory formation. In addition, NO is responsible for some parts of the host response to sepsis and inflammation and contributes to certain disease states. A number of strategies have emerged with regard to a pharmacological control of pathological NO overproductions. This review will discuss these novel therapeutic approaches that may provide new means for clinical medicine.     

Facilitated c-Fos Induction in Mice Deficient for the AMPA Receptor-Associated Protein Ckamp44

The recently identified Cystine-knot containing AMPAR-associated protein (Ckamp44) represents a novel AMPAR-related protein that critically controls AMPAR-mediated currents and short-term plasticity. However, the effects of the lack of this protein at network level are not entirely understood. Here we used c-Fos brain mapping to analyse whether the excitatory/inhibitory balance is altered in the absence of the Ckamp44. We found that Ckamp44^?/? mice treated with an NMDAR antagonist exhibited a very robust c-Fos expression pattern, similar with that seen in mice lacking the GluN2A subunit of NMDAR treated with the same compound. This finding is unexpected, in particular, since Ckamp44 expression is strongest in dentate gyrus granule cells and less abundant in the rest of the brain.     

Imaging cell signalling and movement in development

Imaging is a method of choice to investigate the complex spatio-temporal cellular dynamics and the signalling pathways that control them during development. The ability to tag many proteins in vivo makes it possible to analyse the detailed dynamics of these interactions ranging over several orders of magnitude; from the study of single molecule events on the millisecond and nanometre scale up to the complex three-dimensional behaviour of cells in tissues on the millimetre scale over time periods of hours to days. Great advances are being made in the detailed study of molecular processes using high resolution imaging techniques in transparent samples close to the surface of cells or tissues, where light scattering is minimal. The major challenge is to translate some of these methods to the study of cells and tissues in their native 3D environment. These imaging methods require novel and innovative analysis methods to fully exploit the information available in these data. We will illustrate some of these points in the investigation of the development of the cellular slime mould Dictyostelium discoideum and the study of cell behaviours during gastrulation in the chick embryo.     

Surprising negative association between IgG1 allotype disparity and anti-adalimumab formation: a cohort study

Introduction

The human monoclonal antibody adalimumab is known to induce an anti-globulin response in some adalimumab-treated patients. Antibodies against adalimumab (AAA) are associated with non-response to treatment. Immunoglobulins, such as adalimumab, carry allotypes which represent slight differences in the amino acid sequences of the constant chains of an IgG molecule. Immunoglobulins with particular IgG (Gm) allotypes are racially distributed and could be immunogenic for individuals who do not express these allotypes. Therefore, we investigated whether a mismatch in IgG allotypes between adalimumab and IgG in adalimumab-treated patients is associated with the development of AAA.

Methods

This cohort study consisted of 250 adalimumab-treated rheumatoid arthritis (RA) patients. IgG allotypes were determined for adalimumab and for all patients. Anti-idiotype antibodies against adalimumab were measured with a regular radio immunoassay (RIA), and a newly developed bridging enzyme linked immunosorbent assay (ELISA) was used to measure anti-allotype antibodies against adalimumab. The association between AAA and the G1m3 and the G1m17 allotypes was determined. For differences between groups we used the independent or paired samples t-test, Mann-Whitney test or Chi square/Fisher's exact test as appropriate. To investigate the influence of confounders on the presence or absence of AAA a multiple logistic regression-analysis was used.

Results

Adalimumab carries the G1m17 allotype. No anti-allotype antibodies against adalimumab were detected. Thirty-nine out of 249 patients had anti-idiotype antibodies against adalimumab (16%). IgG allotypes of RA patients were associated with the frequency of AAA: patients homozygous for G1m17 had the highest frequency of AAA (41%), patients homozygous for G1m3 the lowest frequency (10%), and heterozygous patients' AAA frequency was 14% (P = 0.0001).

Conclusions

An allotype mismatch between adalimumab and IgG in adalimumab-treated patients did not lead to a higher frequency of AAA. On the contrary, patients who carried the same IgG allotype as present on the adalimumab IgG molecule, had the highest frequency of anti-adalimumab antibodies compared to patients whose IgG allotype differed from adalimumab. This suggests that the allotype of adalimumab may not be highly immunogenic. Furthermore, patients carrying the G1m17-allotype might be more prone to antibody responses.     

Calcium-induced exocytosis from actomyosin-driven, motile varicosities formed by dynamic clusters of organelles

Varicosities are ubiquitous neuronal structures that appear as local swellings along neurites of invertebrate and vertebrate neurons. Surprisingly little is known about their cell biology. We use here cultured Aplysia neurons and demonstrate that varicosities are motile compartments that contain large clusters of organelles. The content of varicosities propagate along neurites within the plasma membrane “sleeve”, split and merge, or wobble in place. Confocal imaging, retrospective immunolabeling, electron microscopy and pharmacological perturbations reveal that the motility of the varicosities’ organelle content occurs in concert with an actin scaffold and is generated by actomyosin motors. Despite the motility of these organelle clusters within the cytoplasm along the neurites, elevation of the free intracellular calcium concentration within varicosities by trains of action potentials induces exocytosis followed by membrane retrieval. Our observations demonstrate that varicosities formed in the absence of postsynaptic cells behave as “ready to go” prefabricated presynaptic terminals. We suggest that the varicosities’ motility serves to increase the probability of encountering a postsynaptic cell and to rapidly form a functional synapse. Electronic Supplementary Material Supplementary material is available in the online version of this article at These authors contributed equally to the paper.     

Effects of incorporating spatial autocorrelation into the analysis of species distribution data

Aim  Spatial autocorrelation (SAC) in data, i.e. the higher similarity of closer samples, is a common phenomenon in ecology. SAC is starting to be considered in the analysis of species distribution data, and over the last 10 years several studies have incorporated SAC into statistical models (here termed 'spatial models'). Here, I address the question of whether incorporating SAC affects estimates of model coefficients and inference from statistical models.
Methods  I review ecological studies that compare spatial and non-spatial models.
Results  In all cases coefficient estimates for environmental correlates of species distributions were affected by SAC, leading to a mis-estimation of on average c . 25%. Model fit was also improved by incorporating SAC.
Main conclusions  These biased estimates and incorrect model specifications have implications for predicting species occurrences under changing environmental conditions. Spatial models are therefore required to estimate correctly the effects of environmental drivers on species present distributions, for a statistically unbiased identification of the drivers of distribution, and hence for more accurate forecasts of future distributions.     

Modeling of self-organized avascular tumor growth with a hybrid cellular automaton

Pattern formation in multicellular spheroids is addressed with a hybrid lattice-gas cellular automaton model. Multicellular spheroids serve as experimental model system for the study of avascular tumor growth. Typically, multicellular spheroids consist of a necrotic core surrounded by rings of quiescent and proliferating tumor cells, respectively. Furthermore, after an initial exponential growth phase further spheroid growth is significantly slowed down even if further nutrient is supplied. The cellular automaton model explicitly takes into account mitosis, apoptosis and necrosis as well as nutrient consumption and a diffusible signal that is emitted by cells becoming necrotic. All cells follow identical interaction rules. The necrotic signal induces a chemotactic migration of tumor cells towards maximal signal concentrations. Starting from a small number of tumor cells automaton simulations exhibit the self-organized formation of a layered structure consisting of a necrotic core, a ring of quiescent tumor cells and a thin outer ring of proliferating tumor cells.     

Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2- 3Galbeta1-3GalNAc sequences

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)- GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc- Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.      

Novel splice variants associated with one of the zebrafish <Emphasis Type="Italic">dnmt3</Emphasis>genes

Background  

DNA methylation and the methyltransferases are known to be important in vertebrate development and this may be particularly true for the Dnmt3 family of enzymes because they are thought to be the de novo methyltransferases. Mammals have three Dnmt3 genes; Dnmt3a, Dnmt3b, and Dnmt3L, two of which encode active enzymes and one of which produces an inactive but necessary cofactor. However, due to multiple promoter use and alternative splicing there are actually a number of dnmt3 isoforms present. Six different dnmt3 genes have recently been identified in zebrafish.     

Calcium,protease activation,and cytoskeleton remodeling underlie growth cone formation and neuronal regeneration

The cytoarchitecture, synaptic connectivity, and physiological properties of neurons are determined during their development by the interactions between the intrinsic properties of the neurons and signals provided by the microenvironment through which they grow. Many of these interactions are mediated and translated to specific growth patterns and connectivity by specialized compartments at the tips of the extending neurites: the growth cones (GCs). The mechanisms underlying GC formation at a specific time and location during development, regeneration, and some forms of learning processes, are therefore the subject of intense investigation. Using cultured Aplysia neurons we studied the cellular mechanisms that lead to the transformation of a differentiated axonal segment into a motile GC. We found that localized and transient elevation of the free intracellular calcium concentration ([Ca²⁺] _i ) to 200–300 M induces GC formation in the form of a large lamellipodium that branches up into growing neurites. By using simultaneous on-line imaging of [Ca²⁺] _i and of intraaxonal proteolyticactivity, we found that the elevated [Ca²⁺] _i activate proteases in the region in which a GC is formed. Inhibition of the calcium-activated proteases prior to the local elevation of the [Ca²⁺] _i blocks the formation of GCs. Using retrospective immunofluorescent methods we imaged the proteolysis of the submembrane spectrin network, and the restructuring of the cytoskeleton at the site of GC formation. The restructuring of the actin and microtubule network leads to local accumulation of transported vesicles, which then fuse with the plasma membrane in support of the GC expansion.