Sound production by the Shi drum Umbrina cirrosa and comparison with the brown meagre Sciaena umbra: a passive acoustic monitoring perspective.

Sounds produced by the Shi drum Umbrina cirrosa were short trains of pulses with an average pulse period of 180 ms, pulse duration of c. 40 ms and an average peak frequency of 400 Hz; average values of acoustical properties differed from those recorded from the brown meagre Sciaena umbra in previous studies. The present study provides a preliminary tool for discriminating between these two species while conducting passive acoustic monitoring. The potential effects of ontogeny on sound production in both species are discussed and recommendations are made for further research.

DNA immunisation with minimalistic expression constructs.

The low efficacy obtained in large animals makes plasmid-based DNA vaccines commercially unviable. Another concern is the presence of antibiotic resistance markers on virtually all conventional plasmids. Here we describe the use of minimalistic, immunogenically defined gene expression (MIDGE) vectors for DNA vaccination. MIDGE are linear, covalently-closed vectors containing all the essential information for gene expression and none of the non-essential and potentially dangerous plasmid backbone sequences. MIDGE vectors can also be chemically modified on both ends at defined positions allowing targeting of the DNA to specific cell types or cellular compartments. Immunisation of mice with simple and end-modified MIDGE vectors showed that they are efficacious tools to generate and/or manipulate antigen-specific immune responses.

DNA vaccination with linear minimalistic (MIDGE) vectors confers protection against Leishmania major infection in mice.

Immunization protocols based on priming with plasmid DNA and boosting with recombinants of vaccinia virus (rVV) encoding the same antigen offer great promise for the prevention and treatment of many parasitic and viral infections for which conventional vaccination has little or no effect. To overcome some of the potential problems associated to the use of plasmids, we have developed minimalistic, immunogenically defined, gene expression (MIDGE((R))) vectors. These linear vectors contain only the minimum sequence required for gene expression and can be chemically modified to increase the immune response. Here, we demonstrate that MIDGE vectors coding for the LACK antigen confer a highly effective protection against Leishmania infection in susceptible Balb/c mice. Protection is achieved at lower doses of vector compared to conventional plasmids. This efficacy could be greatly improved by the addition of a nuclear localization signal (NLS) peptide to the end of the MIDGE vector. In fact, immunization with two doses of NLS-modified MIDGE conferred similar or even better protection than that achieved by priming with plasmid DNA followed by boosting with rVV. These results demonstrate that MIDGE vectors are a good alternative to plasmid and rVV for immunization.

Receptor dimerization: a key step in chemokine signaling.

Chemokines exert their effects through their interaction with seven transmembrane domain receptors coupled to G-proteins, GPCRs. Such receptor ligation leads to the regulation of numerous activities where chemokines play a key role, including hematopoiesis, T-cell activation, angiogenesis, inflammatory diseases or HIV-1 infection. Here we discuss the molecular mechanisms that underlie chemokine receptor activation. As occurs with other GPCRs, chemokines initiate the signaling cascades by inducing receptor dimerization. This dimerization enables the activation of the JAK/STAT pathway which allows the subsequent triggering of G-protein dependent signaling events. This mechanism provides a new context to explain some of the activities exerted by chemokines and introduces new targets for the development of drugs to fight those diseases were chemokines are implicated, such as inflammation and AIDS.

Chemokine receptor homo- or heterodimerization activates distinct signaling pathways.

Chemokine receptors of both the CC and CXC families have been demonstrated to undergo a ligand-mediated homodimerization process required for Ca2+ flux and chemotaxis. We show that, in the chemokine response, heterodimerization is also permitted between given receptor pairs, specifically between CCR2 and CCR5. This has functional consequences, as the CCR2 and CCR5 ligands monocyte chemotactic protein-1 (MCP-1) and RANTES (regulated upon activation, normal T cell-expressed and secreted) cooperate to trigger calcium responses at concentrations 10- to 100-fold lower than the threshold for either chemokine alone. Heterodimerization results in recruitment of each receptor-associated signaling complex, but also recruits dissimilar signaling path ways such as G(q/11) association, and delays activation of phosphatidyl inositol 3-kinase. The consequences are a pertussis toxin-resistant Ca2+ flux and trig gering of cell adhesion rather than chemotaxis. These results show the effect of heterodimer formation on increasing the sensitivity and dynamic range of the chemokine response, and may aid in understanding the dynamics of leukocytes at limiting chemokine concentrations in vivo.

HIV-1 infection through the CCR5 receptor is blocked by receptor dimerization.

The identification of the chemokine receptors as receptors for HIV-1 has boosted interest in these molecules, raising expectations for the development of new strategies to prevent HIV-1 infection. The discovery that chemokines block HIV-1 replication has focused attention on identifying their mechanism of action. Previous studies concluded that this inhibitory effect may be mediated by steric hindrance or by receptor down-regulation. We have identified a CCR5 receptor-specific mAb that neither competes with the chemokine for binding nor triggers signaling, as measured by Ca(2+) influx or chemotaxis. The antibody neither triggers receptor down-regulation nor interferes with the R5 JRFL viral strain gp120 binding to CCR5, but blocks HIV-1 replication in both in vitro assays using peripheral blood mononuclear cells as HIV-1 targets, as well as in vivo using human peripheral blood mononuclear cell-reconstituted SCID (severe combined immunodeficient) mice. Our evidence shows that the anti-CCR5 mAb efficiently prevents HIV-1 infection by inducing receptor dimerization. Chemokine receptor dimerization also is induced by chemokines and is required for their anti-HIV-1 activity. In addition to providing a molecular mechanism through which chemokines block HIV-1 infection, these results illustrate the prospects for developing new tools that possess HIV-1 suppressor activity, but lack the undesired inflammatory side effects of the chemokines.

The chemokine SDF-1alpha triggers CXCR4 receptor dimerization and activates the JAK/STAT pathway.

The chemokine stromal cell-derived factor (SDF-1alpha), the ligand for the CXCR4 receptor, induces a wide variety of effects that include calcium mobilization, chemotactic responses, bone marrow myelopoiesis, neuronal patterning, and prevention of HIV-1 infection. Nonetheless, little is known of the biochemical pathways required to achieve this variety of responses triggered after receptor-chemokine interaction. We developed a set of monoclonal antibodies that specifically recognize the CXCR4 receptor and used them to identify the signaling pathway activated after SDF-1alpha binding in human T cell lines. Here we demonstrate that SDF-1alpha activation promotes the physical association of Galpha(i) with the CXCR4. Furthermore, within seconds of SDF-1alpha activation, the CXCR4 receptor becomes tyrosine phosphorylated through the activation and association with the receptor of JAK2 and JAK3 kinases. After SDF-1alpha binding, JAK2 and JAK3 associate with CXCR4 and are activated, probably by transphosphorylation, in a Galpha(i)-independent manner. This activation enables the recruitment and tyrosine phosphorylation of several members of the STAT family of transcription factors. Finally, we have also observed SDF-1alpha-induced activation and association of the tyrosine phosphatase Shp1 with the CXCR4 in a Galpha(i)-dependent manner. As occurs with the cytokine receptors in response to cytokines, the CXCR4 undergoes receptor dimerization after SDF-1alpha binding and is a critical step in triggering biological responses. We present compelling evidence that the chemokines signal through mechanisms similar to those activated by cytokines.

Characterization of RANTES- and aminooxypentane-RANTES-triggered desensitization signals reveals differences in recruitment of the G protein-coupled receptor complex.

The trafficking of lymphocyte populations is a complex process controlled by a vast array of molecules. In this process, cells must be able to sense small changes in chemoattractant gradients. Migration through a chemotactic gradient probably employs an on-off mechanism in which chemokine receptor desensitization, internalization, and recycling may be important steps. This multistep process requires the coordinated action of many factors, including G protein-coupled receptor kinases, arrestins, clathrin, and GTP-hydrolyzing proteins such as dynamin. In this report, we show that RANTES and its derivative, aminooxypentane (AOP)-RANTES, a potent RANTES antagonist as well as an inhibitor of HIV-1 infection, both promote CCR5 desensitization involving G protein-coupled receptor kinases-2 and beta-arrestin equally well. An important difference between the two molecules is that (AOP)-RANTES is more efficient than RANTES in promoting Ser/Thr phosphorylation of the receptor and association of G protein-coupled receptor kinases-2, beta-arrestin, and clathrin to the CCR5. After stimulation with either ligand, we observe rapid, transient association of dynamin to CCR5, implicating this protein in receptor sensitization, but this association is faster and longer-lasting following (AOP)-RANTES stimulation. In summary, we show that chemokine receptor internalization takes place through the formation of clathrin vesicles and involves dynamin activity. We provide compelling evidence that the differences between RANTES and (AOP)-RANTES in G alpha i activation condition subsequent signaling events, including internalization and receptor recycling.

The chemokine monocyte chemoattractant protein-1 induces functional responses through dimerization of its receptor CCR2.

Cytokines interact with hematopoietin superfamily receptors and stimulate receptor dimerization. We demonstrate that chemoattractant cytokines (chemokines) also trigger biological responses through receptor dimerization. Functional responses are induced after pairwise crosslinking of chemokine receptors by bivalent agonistic antichemokine receptor mAb, but not by their Fab fragments. Monocyte chemoattractant protein (MCP)-1-triggered receptor dimerization was studied in human embryonic kidney (HEK)-293 cells cotransfected with genes coding for the CCR2b receptor tagged with YSK or Myc sequences. After MCP-1 stimulation, immunoprecipitation with Myc-specific antibodies revealed YSK-tagged receptors in immunoblotting. Receptor dimerization also was validated by chemical crosslinking in both HEK-293 cells and the human monocytic cell line Mono Mac 1. Finally, we constructed a loss-of-function CCR2bY139F mutant that acted as a dominant negative, blocking signaling through the CCR2 wild-type receptor. This study provides functional support for a model in which the MCP-1 receptor is activated by ligand-induced homodimerization, allowing discussion of the similarities between bacterial and leukocyte chemotaxis.

Similarities and differences in RANTES- and (AOP)-RANTES-triggered signals: implications for chemotaxis.

Chemokines are a family of proinflammatory cytokines that attract and activate specific types of leukocytes. Chemokines mediate their effects via interaction with seven transmembrane G protein-coupled receptors (GPCR). Using CCR5-transfected HEK-293 cells, we show that both the CCR5 ligand, RANTES, as well as its derivative, aminooxypentane (AOP)- RANTES, trigger immediate responses such as Ca2+ influx, receptor dimerization, tyrosine phosphorylation, and Galphai as well as JAK/STAT association to the receptor. In contrast to RANTES, (AOP)-RANTES is unable to trigger late responses, as measured by the association of focal adhesion kinase (FAK) to the chemokine receptor complex, impaired cell polarization required for migration, or chemotaxis. The results are discussed in the context of the dissociation of the late signals, provoked by the chemokines required for cell migration, from early signals.

The chemokine monocyte chemotactic protein 1 triggers Janus kinase 2 activation and tyrosine phosphorylation of the CCR2B receptor.

The chemokines are a growing family of low m.w., 70- to 80-residue proinflammatory cytokines that operate by interacting with G protein-coupled receptors. Chemokines are involved in cell migration and in the activation of specific leukocyte subsets. Using the Mono Mac 1 monocytic cell line, we show that monocyte chemotactic protein 1 (MCP-1) triggers activation of the Janus kinase 2 (JAK2)/STAT3 pathway and CCR2 receptor tyrosine phosphorylation. Both Ca2+ mobilization and cell migration are blocked in Mono Mac 1 cells by tyrphostin B42, a specific JAK2 kinase inhibitor. Within seconds of MCP-1 activation, JAK2 phosphorylates CCR2 at the Tyr139 position and promotes JAK2/STAT3 complex association to the receptor. This MCP-1-initiated phosphorylation and association to JAK2 is also observed in CCR2B-transfected HEK293 cells. In contrast, when a CCR2B Tyr139Phe mutant is expressed in HEK293 cells, it is not phosphorylated in tyrosine and triggers neither JAK2/STAT3 activation nor Ca2+ mobilization in response to MCP-1. These results implicate the tyrosine kinase pathway in early chemokine signaling, suggesting a key role for this kinase in later events.

The relationship between skin maturation and electrical skin impedance.

When performing electrophysiological testing, high electrical impedance values are sometimes found in neonates. Since excessive impedance can invalidate test results, a study was conducted to delineate the relationship between skin maturation and electrical skin impedance. This study investigated the skin impedance in 72 infants ranging from 196 to 640 days of age from conception. Regression analyses demonstrated a significant relationship between impedance and age, with the highest impedance centered around full-term gestation with values falling precipitously at time points on either side. Clinically, impedance values fall to normal levels at approximately four months following full-term gestation. Skin impedance values are low in premature infants, but rapidly increase as the age approaches that of full-term neonates. Low impedance values in premature infants are attributed to greater skin hydration which results from immature skin conditions such as 1) thinner epidermal layers particularly at the transitional and cornified layers; 2) more blood flow to the skin; and 3) higher percentage of water composition. These factors facilitate the diffusion of water vapor through the skin. As the physical barrier to skin water loss matures with gestational age, the skin impedance reaches a maximum value at full term neonatal age. After this peak, a statistically significant inverse relationship exists between electrical skin impedance and age in the first year of life. This drop in skin impedance is attributed to an increase in skin hydration as a result of the greater functional maturity of eccrine sweat glands.

Brain and ocular abnormalities in infants with in utero exposure to cocaine and other street drugs.

We describe 10 infants with developmental delay and congenital cerebral anomalies who were found to have had in utero exposure to vasoactive drugs. Nine infants had ophthalmological abnormalities; these included strabismus, nystagmus, and/or hypoplastic optic discs. Six mothers used cocaine, one used cocaine and heroin, one used only heroin, one used amphetamine, and one used phenylpropanolamine. Each of these cerebral anomalies (agenesis of the corpus callosum, septo-optic dysplasia, schizencephaly, hydranencephaly, congenital hydrocephalus, porencephaly, and cerebral infarctions) can be attributed to insults at different stages of development. There appears to be a relationship between the time of prenatal drug exposure and the type of cerebral anomaly, evoking malformations, disruptions, or fetal strokes. Since many or possibly all of these anomalies are thought to have a vascular origin, it seems appropriate to implicate prenatal exposure to vasoactive drugs.

Vascular microdissection in strabismus surgery.

Microdissection of the muscular vessels allows recessions, resections, or transpositions of the recti muscles without interrupting the blood flow to the corresponding anterior ciliary arteries. This avoids postoperative impaired blood flow to the anterior segment and may play a role in preventing anterior segment ischemia syndrome. The use of a fine-hooked cannula and microscissors, in addition to the usual strabismus surgery instruments, is described.

Congenital generalized fibromatosis.

Congenital fibromatosis is a rare form of fibroblastic proliferation, which may appear similar to fibrosarcoma; it is multicentric, involving soft tissues, viscera, and bones. The larger soft tissue tumors are very hemorrhagic and necrotic, and they may contain calcifications. Because these characteristics are also present in the more common hemangiomas of infancy, congenital fibromatosis can be diagnosed erroneously as hemangioma. We report a case of congenital generalized fibromatosis erroneously diagnosed initially as hemangioma. The multicentric tumor was aggressive, ultimately resulting in death.

Lacrimal tube repositioning: technique and ultrasonic assessment.

A section of migrated lacrimal silicone tubing was repositioned in a 7-year-old patient by dilating the lacrimal passage with hyaluronate solution and pushing the tube back into the nasal fossa with a probe. The position of the tube was postoperatively confirmed by ultrasonographic studies.

Timed biomicroscopy of rabbit corneal wound repair.

Central, full-thickness excisional wounds were made in rabbit corneas to evaluate the biomicroscopic changes during the scarring process. Within the first minutes, the wound is closed with a fibrin clot. A timely microscopic study was performed in 10 rabbits (group A) during the first minutes after surgery to study the morphological changes in fibrin clot formation. A lid speculum was used for lid retraction to continuously expose the eyes. The fibrin clot formed in the first minutes between the wound edges and grew progressively. Subsequent breaks and reconstruction of the clot occurred. With this method, a permanent wound closure did not occur in any of the 10 eyes during the first 90 min. Twenty more rabbits (group B) were used to study the wound repair in more physiological conditions without lid retraction. Microphotographs were obtained at 6 and 12 h, and at 1, 2, 7, 30 and 90 days after surgery. Three months after the procedure, the appearance of the cornea was stable, with a thick leukoma occupying the lesioned area.