Dll4-Notch signaling determines the formation of native arterial collateral networks and arterial function in mouse ischemia models.

Arteriogenesis requires growth of pre-existing arteriolar collateral networks and determines clinical outcome in arterial occlusive diseases. Factors responsible for the development of arteriolar collateral networks are poorly understood. The Notch ligand Delta-like 4 (Dll4) promotes arterial differentiation and restricts vessel branching. We hypothesized that Dll4 may act as a genetic determinant of collateral arterial networks and functional recovery in stroke and hind limb ischemia models in mice. Genetic loss- and gain-of-function approaches in mice showed that Dll4-Notch signaling restricts pial collateral artery formation by modulating arterial branching morphogenesis during embryogenesis. Adult Dll4(+/-) mice showed increased pial collateral numbers, but stroke volume upon middle cerebral artery occlusion was not reduced compared with wild-type littermates. Likewise, Dll4(+/-) mice showed reduced blood flow conductance after femoral artery occlusion, and, despite markedly increased angiogenesis, tissue ischemia was more severe. In peripheral arteries, loss of Dll4 adversely affected excitation-contraction coupling in arterial smooth muscle in response to vasopressor agents and arterial vessel wall adaption in response to increases in blood flow, collectively contributing to reduced flow reserve. We conclude that Dll4-Notch signaling modulates native collateral formation by acting on vascular branching morphogenesis during embryogenesis. Dll4 furthermore affects tissue perfusion by acting on arterial function and structure. Loss of Dll4 stimulates collateral formation and angiogenesis, but in the context of ischemic diseases such beneficial effects are overruled by adverse functional changes, demonstrating that ischemic recovery is not solely determined by collateral number but rather by vessel functionality.

Endless forms of endless formation - The morphogenesis of organisms and scientific objects.

The present article proceeds from the premises that living forms and abstract formalization come into being by similar mechanisms (e.g., random variation, selection, conventions) and have similar properties (e.g., semiosis, stasis and complexity). These convergences justify the comparative analysis of form's development, evolution and action in both fields. Here we shall focus on the notion of "endless forms" advanced by Darwin's seminal work in evolutionary biology "On The Origin of Species" to discuss the various ways in which it relates to biological formation. I shall explore the idea of "infinitude of evolved forms" through the lens of the five connotations of the word "endless" provided by the Merriam-Webster Thesaurus dictionary, which are: perpetual; incomputable; manifold; unfinished; steady. From each synonym chosen, a new iteration of dictionary search was made to produce a list of terms that are used in the reviewed literature to describe biological morphogenetic features, which are respectively: reproducible, unpredictable, additive, undetermined, the end of their own formation. In conclusion, I propose a tentative mapping between each of these five connotations and the biological processes at work in their making, which are, respectively: 1) copying organic information; coding organic signs; manufacturing organic meaning 2) natural variation, natural selection, natural conventions; 3) multilevel organization, differentiation/development, complexity; 4) ambiguity, degeneracy, semiotic thresholds; 5) homeostasis, autopoiesis, codepoiesis. The processes discussed here gained salience as developments, additions, or nuances to Darwin's original theory. It must be noted that, even though the discussion is mainly framed by Code Biology as a source of conceptualization, inputs from a wide range of theoretical perspectives will be given emphasis when suitable.

Autolytic Mycobacterium leprae Hsp65 fragments may act as biological markers for autoimmune diseases.

Investigating the proteolytic activity of the recombinant Mycobacterium leprae Heat Shock Protein of 65 kDa (rHsp65), chaperonin 2 (cpn2), we observed that it displays high instability. The fragmentation process starts at the C-terminus followed by progressive degradation of the N-terminus, which leads to a stable fragment comprising the middle region of the molecule. Urea was able to prevent autolysis, probably due to its denaturing action, while EDTA increased degradation levels indicating the need for metal ions. Peptides originated from autolysis were purified and analyzed by mass spectrometry, generating a continuous map. Since the bacteria and mammalian Hsp60 are known to be targets of the immune response and have been implicated in autoimmune diseases and chronic inflammation, the in vivo effect of rHsp65 peptides was evaluated in the spontaneous Systemic Lupus Erythematosus (SLE) model developed by the (NZB/NZW)F(1) mouse hybrids, and their individual anti-rHsp65 IgG2a/IgG1 antibody titer ratio was determined. The results showed orientation toward a T(H)1 responsiveness, and the treatment with the rHsp65 peptides diminished the environmental variance of the survival time of treated animals. These results outline the fact that environmental factors may also act through the modified stability expression of Heat Shock Proteins intervening during autoimmune processes.

Two theories of action - Edelman's Neuronal group Selection and the Poetics of Paul Valéry.

Motivated by two imperatives as they are framed in Code Biology - mechanism and actualization - we have turned to other attempts of modeling life at work. Here, we present two theories devoted to minds in action - one explains neuronal function, and the other dissects poetic crafting. Neuronal networks activation and poetic composition, respectively, are seen as the selection of specific connective patterns of either neurons or words, in action. Gerald Edelman, as a scientist, has generalized the Darwinian ideas of variation and selection to the cellular level in his "Sciences of Recognition", a broader theoretical framework that includes the "Theory of Neuronal Group Selection" (TGNS) analyzed here. Paul Valéry, as a poet, has reconciled inspiration and technique in what he has called "works of the mind", the creative processes mediated by sensing and making sense, in the "Poetic Theory" we present here he advances the mechanisms of artistic composition. We have identified the main ideas conveyed in these two theories, i.e., variation and selection, integration and differentiation, ambiguity and degeneracy, binding and blending, stasis and semiosis, by pairing and comparing textual fragments from the authors. We show that TGNS and the Theory of Poetic Action reconcile Sciences and Arts by recognizing that Natural Selection is a mechanism implied by formative acts in both scenarios and discuss to which extent Natural Convention - the main contribution of Code Biology - is integrated by the two thinkers.

Word games and molecular recognition - The creative control of endothelial cell function.

In this paper we postulate that figures of language and structural features, which are characteristic of poetic writings and word games, have direct correlates in the cell/cell and cell/matrix recognition processes. We shall discuss the particular example of endothelial cells engagement into angiogenesis (the making of new blood vessels in adult vertebrates)-an activity that requires intense extracellular matrix (ECM) remodeling and Cell Adhesion Molecules (CAMs) rearrangement-to argue that angiogenesis controls are organized into semiotic dimensions. CAMs attach cells to the ECM; they are mainly composed of integrins (transmembrane receptors) that bind selectively to different ECM components. After ECM binding, the cytoplasmic tails of integrins within the cell will begin to interact with a wide range of recruited factors that, in turn, regulate integrin clustering in the cell membrane. These recruited factors also activate signaling pathways that link activated integrins to later microfilament system remodeling during cell migration. Ultimately CAMs work as functional protein networks-as the adaptor molecules in an organic code, the Adhesion Code-controlling cell migration/proliferation transition through the continual rearrangement of both ECM adhesion and Actin polymerization. Additionally, orchestrating the transition from clonal growth to differentiation, the interaction between CAMs and ECM components will trigger specific Signal Transduction pathways. We shall examine some attempts to conceptualize "somatic cell function" in the recent specialized literature, which introduces the notion of hierarchic organization into levels i.e. molecular, sub-cellular and cellular and describes an informational flow of increasing complexity versus decreasing number of entities through the levels. Beyond the syntactic level-the specific recognition of discrete ECM motifs by integrin heterodimers-we shall examine the semantic and pragmatic levels at which the composition and architecture of multimolecular complexes will dictate cell recognition events and cell fate decisions. These higher-level codes will be compared to the dynamics of the word games of Lewis Carroll, e.g. "doublets" and "magic squares." In such creative games words are subjected to synthetic transformations that have to conform to semantic rules, but are ultimately constrained by meaning, as concrete pragmatics. This gives us a model for analyzing angiogenesis control as a word game.

Sugar boost: when ribose modifications improve oligonucleotide performance.

Since the first antisense oligonucleotides were described, synthetic oligonucleotides have been used to modulate gene expression for numerous research and therapeutic purposes over the last 30 years. In order to improve the performance of oligonucleotides in therapeutic applications, various chemical modifications of nucleotides have been developed and applied to antisense, antigen, aptamer and short interfering RNA (siRNA) therapeutics. For example, backbone substitution of phosphodiester bonds to create DNA phosphorothioates and 4'-thio RNA analogs, increases resistance to nuclease degradation. Modifications to improve the activity of oligonucleotides by 2'-O-modification of the ribose sugar, and the systematic approaches used to evaluate the potential of mixed oligonucleotides, are discussed in this review. Although structural data from crystallization studies appear to confirm observed gains in thermodynamic stability with 2'-O-modifications, no simple rules can be drawn to predict the overall effect of individual modifications for the rational design of mixed oligonucleotides.

Characterization of urinary metabolites from four synthetic bradykinin potentiating peptides (BPPs) in mice.

BPPs have been identified in the venom of the Bothrops jararaca snake, or deduced from precursor proteins expressed either in the venom gland or in the brain of the snake. Their potentiating activity on bradykinin (Bk) is assumed to occur through a somatic angiotensin-converting enzyme (sACE) inhibitory mechanism. We have demonstrated that synthetic BPPs show remarkable functional differences, despite their high amino acid sequence similarities. Recently, we demonstrated that BPP-10c, after i.p. administration, was found in its intact form and in the form of a unique metabolite (des-Pro(10) BPP-10c) in mouse urine. Given this finding, we selected a number of BPPs with different structure-activities - BPP-5a (

Peptides derived from plasma proteins released by bothropasin, a metalloprotease present in the Bothrops jararaca venom.

Viperid snake venoms contain proteases that affect hemostasis by degrading important proteins such as those that participate in the coagulation cascade. The Bothrops jararaca venom presents as its main components metallo and serine proteases, which comprise around 65% of the venom composition. Bothropasin is a hemorrhagic metalloprotease from the B. jararaca venom which causes disruption of the basement membrane of the vascular endothelium, resulting in bleeding. Although the bothropasin ability to degrade plasmatic and extracellular matrix proteins in vitro has been described, the primary sequence of the released peptides is unknown. This research study presents the peptide identification from both fibrinogen and fibronectin, generated by bothropasin proteolytic activity. Among the fibrinogen derived peptides identified by mass spectrometry, analogous of endogenous products like the fibrinopeptides A and B were found, as well as other sequences described in the literature with vasoactive or antiangiogenic properties. A series of peptides derived from fibronectin by the action of bothropasin were described, and for most of them no biological activity has been described. However, exceptionally a peptide that is known as a bond site for B cells was found. This study indicates that, beyond to the degradation of human proteins, bothropasin can generate bioactive peptides, which may participate in the envenoming process by Bothrops snakes. Also important, the knowledge of the formed peptides, based on the cleavage sites of the hydrolyzed proteins, provided the opportunity to study the primary specificity of bothropasin.

The use of synthetic oligonucleotides as protein inhibitors and anticode drugs in cancer therapy: accomplishments and limitations.

The function of gene products can be altered at many levels, including the mutation of gene sequence and the change in steady state levels of mRNA and/or protein by various mechanisms. The cumulative malfunction of specific gene products underlies many pathological conditions such as the multi-step and multi-cause acquisition of cancer. Here we discuss two oligonucleotide-based strategies in which these compounds target defective gene products acting either as antiprotein or anticode agents. The SELEX technique (systematic evolution of ligands by exponential enrichment) is an antiprotein approach in which nuclease-resistant DNA or RNA aptamers are selected by their ability to bind their protein targets with high affinity and specificity of the same range as antibodies. Such inhibitors were previously evolved against a great variety of targets, including receptors, growth factors and adhesion molecules implicated in the genesis of some kinds of cancer. Moreover, some results have already been obtained in animal models. The antigene technology interferes with earlier steps in the information flow leading from gene to protein. In this approach selective gene silencing is provided by the formation of stable and specific complexes between triplex forming molecules and their DNA targets. The feasibility of this strategy as well as a molecular mechanism for the action of antigene oligonucleotides has been demonstrated in cellular systems and in vivo. The use of oligonucleotide drugs (of either the antiprotein or the anticode type) as a viable approach to cancer therapy is limited by some common problems that will be discussed.

Histone H1 is phosphorylated in non-replicating and infective forms of Trypanosoma cruzi.

The nuclear structure changes during the differentiation from growing to infective stages of Trypanosoma cruzi. As histone modifications have been correlated with structural and functional changes of chromatin, we investigated whether histones in T. cruzi are modified during the life cycle of this protozoan parasite. We found that histone H1 isolated from proliferating forms (epimastigotes) and from differentiated/infective forms (trypomastigotes) have a distinct migrating pattern in Triton-acetic acid-urea gel electrophoresis. While epimastigotes contain predominantly a fast migrating form, a slow migrating band is prominent in trypomastigotes. By metabolically labeling the cells with radioactive phosphate, we demonstrated that the slow migrating histone H1 band is phosphorylated, and that after alkaline phosphatase treatment, it migrates as the fast form. Parasites arrested at the onset of the S phase of the cell cycle with hydroxyurea (HU) also predominantly have the phosphorylated form of histone H1, suggesting that phosphorylation occurs in non-replicating stages of T. cruzi. We also found that the phosphorylated histone H1 is more weakly associated with the chromatin, being preferentially released at 150 mM NaCl. Therefore, histone H1 phosphorylation varies during the life cycle of T. cruzi, and might be related to changes in the chromatin structure.

Are there epigenetic controls in Trypanosoma cruzi?

RNA interference (RNAi) is defined as the mechanism through which double-stranded RNA (dsRNA) triggers degradation of homologous transcripts. Besides providing an invaluable tool to downregulate gene expression in a variety of organisms, it is now evident that RNAi acts beyond the cytoplasm and is involved in a variety of gene-silencing phenomena in the nucleus. In the present work we review the current status of the knowledge about RNAi in protozoan parasites that belong to the Trypanosoma genus and have medical relevance. While RNAi was first discovered in Trypanosoma brucei, it became evident that other members of the same genus of organisms, namely Trypanosoma cruzi, does not possess RNAi, probably due to the lack of Ago protein analogs in their genomes. We will discuss the genome organization of Trypanosoma cruzi and propose that the absence of both RNAi and gene promoters is symptomatic of alternative epigenetic controls in this parasite orchestrated by parasite-host interactions. Whereas in Trypanosoma brucei, RNAi and other epigenetic controls dictate alternative transcriptional programs critical for virulence.

L-arginine signalling potential in the brain: the peripheral gets central.

L-arginine is a basic amino acid that has versatile metabolic roles, being involved in the generation of a wide range of biologically active intermediates such as nitric oxide (NO), polyamines, creatine and L-amino acids [1]. Because the levels of L-arginine reflect a metabolic crossroads, the mechanisms of its synthesis and degradation in peripheral tissues are very well described. However, there is an increasing amount of data also implicating this amino acid as a mediator of central nervous system activities and those are not yet fully understood. Here we shall summarize the tissue-specific pathways controlling L-arginine intracellular and blood levels and also the emerging evidence pointing to a role of this metabolite in the central regulation of diverse physiological processes, as blood pressure control and inflammatory response. As a conclusion we shall discuss the advantages of targeting L-arginine metabolism over other NO donors, as a general strategy to correct NO deficiency related diseases and discuss a few new patents recently deposited which take into account the rational perspective outlined in the present review.

Administration of M. leprae Hsp65 interferes with the murine lupus progression.

The heat shock protein [Hsp] family guides several steps during protein synthesis, are abundant in prokaryotic and eukaryotic cells, and are highly conserved during evolution. The Hsp60 family is involved in assembly and transport of proteins, and is expressed at very high levels during autoimmunity or autoinflammatory phenomena. Here, the pathophysiological role of the wild type [WT] and the point mutated K(409)A recombinant Hsp65 of M. leprae in an animal model of Systemic Lupus Erythematosus [SLE] was evaluated in vivo using the genetically homogeneous [NZBxNZW]F(1) mice. Anti-DNA and anti-Hsp65 antibodies responsiveness was individually measured during the animal's life span, and the mean survival time [MST] was determined. The treatment with WT abbreviates the MST in 46%, when compared to non-treated mice [p<0.001]. An increase in the IgG2a/IgG1 anti-DNA antibodies ratio was also observed in animals injected with the WT Hsp65. Incubation of BALB/c macrophages with F(1) serum from WT treated mice resulted in acute cell necrosis; treatment of these cells with serum from K(409)A treated mice did not cause any toxic effect. Moreover, the involvement of WT correlates with age and is dose-dependent. Our data suggest that Hsp65 may be a central molecule intervening in the progression of the SLE, and that the point mutated K(409)A recombinant immunogenic molecule, that counteracts the deleterious effect of WT, may act mitigating and delaying the development of SLE in treated mice. This study gives new insights into the general biological role of Hsp and the significant impact of environmental factors during the pathogenesis of this autoimmune process.

Is there a rational approach for increasing drug specificity? Considerations on CNS target choice and validation.

The description of mental illness states brings into light a referential paradox on the absence of grounds for normality. Furthermore, the semiology itself poses a problem throughout the intricate consensual relations between psychiatrists. New molecules with activity on the CNS are ever more specific as to molecular cognitive capabilities, reaching limits of individual genetic variability. Cultural mechanisms of neuronal adaptation also contribute significantly to representations and its correlation with feelings. Neuropeptides increase excitability in various different brain regions, with networks underlying optimal behaviour patterns. Therefore, the sole specification of target molecules yet does not lead directly to specific results, as insights from a systematic approach should conceal. Current validation methods generate insufficient data for discriminating successful treatable candidates. Instead of regarding the heuristics of empirically classified disease models, a new tendency to compromise scientia rationale with technical capabilities should be regarded. Some of the drugs that have obtained patents recently will be discussed in the framework of their rational and actual specificity. The molecular basis underlining function will be contrasted with an alternative approach, namely: how functional organization constrains molecular action. The categories comprising neurogenarative pathologies at one hand and the mood disorders at the other hand will be analysed separately as the procedures guiding drug design in each case seem to be different.

Neuronal differentiation of P19 embryonal carcinoma cells modulates kinin B2 receptor gene expression and function.

Kinins are vasoactive oligopeptides generated upon proteolytic cleavage of low and high molecular weight kininogens by kallikreins. These peptides have a well established signaling role in inflammation and homeostasis. Nevertheless, emerging evidence suggests that bradykinin and other kinins are stored in the central nervous system and may act as neuromediators in the control of nociceptive response. Here we show that the kinin-B2 receptor (B2BKR) is differentially expressed during in vitro neuronal differentiation of P19 cells. Following induction by retinoic acid, cells form embryonic bodies and then undergo neuronal differentiation, which is complete after 8 and 9 days. Immunochemical staining revealed that B2BKR protein expression was below detection limits in nondifferentiated P19 cells but increased during the course of neuronal differentiation and peaked on days 8 and 9. Measurement of [Ca(2+)](i) in the absence and presence of bradykinin showed that most undifferentiated cells are unresponsive to bradykinin application, but following differentiation, P19 cells express high molecular weight neurofilaments, secrete bradykinin into the culture medium, and respond to bradykinin application with a transient increase in [Ca(2+)](i). However, inhibition of B2BKR activity with HOE-140 during early differentiation led to a decrease in the size of embryonic bodies formed. Pretreatment of differentiating P19 cells with HOE-140 on day 5 resulted in a reduction of the calcium response induced by the cholinergic agonist carbamoylcholine and decreased expression levels of M1-M3 muscarinic acetylcholine receptors, indicating crucial functions of the B2BKR during neuronal differentiation.

Chromosome localization changes in the Trypanosoma cruzi nucleus.

Chromosome localization in the interphase nuclei of eukaryotes depends on gene replication and transcription. Little is known about chromosome localization in protozoan parasites such as trypanosomes, which have unique mechanisms for the control of gene expression, with most genes being posttranscriptionally regulated. In the present study, we examined where the chromosomes are replicated in Trypanosoma cruzi, the agent of Chagas' disease. The replication sites, identified by the incorporation of 5-bromodeoxyuridine, are located at the nuclear periphery in proliferating epimastigote forms in the early S phase of the cell cycle. When the S phase ends and cells progress through the cell cycle, 5-bromodeoxyuridine labeling is observed in the nuclear interior, suggesting that chromosomes move. We next monitored chromosome locations in different stages of the cell cycle by using a satellite DNA sequence as a probe in a fluorescence in situ hybridization assay. We found two distinct labeling patterns according to the cell cycle stage. The first one is seen in the G(1) phase, in hydroxyurea-arrested epimastigotes or in trypomastigotes, which are differentiated nondividing forms. In all of these forms the satellite DNA is found in dots randomly dispersed in the nucleus. The other pattern is found in cells from the S phase to the G(2) phase. In these cells, the satellite DNA is found preferentially at the nuclear periphery. The labeling at the nuclear periphery disappears only after mitosis. Also, DNA detected with terminal deoxynucleotidyl transferase is found distributed throughout the nuclear space in the G(1) phase but concentrated at the nuclear periphery in the S phase to the G(2) phase. These results strongly suggest that T. cruzi chromosomes move and, after entering the S phase, become constrained at the nuclear periphery, where replication occurs.