Mutually Independent Cyclic AMP and Sodium Responses to Nerve Growth Factor in Embryonic Chick Dorsal Root Ganglia

Abstract: Chick embryo dorsal root ganglia display a rapid and transient rise in their cyclic AMP content when presented with nerve growth factor. These ganglia also depend on nerve growth factor for control of their intracellular Na⁺ and K⁺ levels. A sequential relationship between the cyclic AMP and Na⁺ responses is not readily apparent. Incubation of chick sensory ganglia in a sodium-free medium does not prevent the cyclic AMP response to nerve growth factor from occurring. When ganglia are first incubated with ouabain for 6 h, presentation of nerve growth factor elicits a cyclic AMP response, but no Na⁺ response. The cyclic AMP response therefore does not depend on the Na⁺ environment. An initial presentation of nerve growth factor to the ganglia for 30 min, followed by its withdrawal and subsequent re-administration at different intervals over several hours failed to result in a second cyclic AMP response. Nevertheless, the expected Na⁺ behaviors were still observed. Dibutyryl cyclic AMP is capable of eliciting a cyclic AMP response in chick sensory ganglia after 6 h of nerve growth factor deprivation. When both agents were presented simultaneously to the ganglia, only a single cyclic AMP response was obtained, corresponding in time to the response elicited by dibutyryl cyclic AMP alone-indicating that this drug acts on the NGF-sensitive cells. At the same time dibutyryl cyclic AMP alone failed to result in a Na⁺ response, leading one to conclude that the cyclic AMP response to nerve growth factor is truly not mediating the Na⁺ response. Additional support for the mutual independence of these two short-latency responses is provided by the apparent inability of nerve growth factor to cause a cyclic AMP response in chick embryo sympathetic ganglia, another traditional target for the factor, which is capable of displaying a Na⁺ response.     

The specificity of SNARE pairing in biological membranes is mediated by both proof-reading and spatial segregation

Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins mediate organelle fusion in the secretory pathway. Different fusion steps are catalyzed by specific sets of SNARE proteins. Here we have used the SNAREs mediating the fusion of early endosomes and exocytosis, respectively, to investigate how pairing specificity is achieved. Although both sets of SNAREs promiscuously assemble in vitro, there is no functional crosstalk. We now show that they not only colocalize to overlapping microdomains in the membrane of early endosomes of neuroendocrine cells, but also form cis-complexes promiscuously, with the proportion of the different complexes being primarily dependent on mass action. Addition of soluble SNARE molecules onto native membranes revealed preference for cognate SNAREs. Furthermore, we found that SNAREs are laterally segregated at endosome contact sites, with the exocytotic synaptobrevin being depleted. We conclude that specificity in endosome fusion is mediated by the following two synergistically operating mechanisms: (i) preference for the cognate SNARE in 'trans' interactions and (ii) lateral segregation of SNAREs, leading to relative enrichment of the cognate ones at the prospective fusion sites.     

Two novel dermonecrotic toxins LiRecDT4 and LiRecDT5 from brown spider (Loxosceles intermedia) venom: from cloning to functional characterization

Loxoscelism (the condition produced by the bite of brown spiders) has been reported worldwide, but especially in warmer regions. Clinical manifestations include skin necrosis with gravitational spreading while systemic loxoscelism may include renal failure, hemolysis and thrombocytopenia. The venom contains several toxins, of which the best biochemically and biologically studied is the dermonecrotic toxin, a phospholipase-D. Purified toxin induces cutaneous and systemic loxoscelism, especially necrotic lesions, hematological disturbances and renal failure. Herein, we describe cloning, heterologous expression and purification of two novel dermonecrotic toxins: LiRecDT4 and LiRecDT5. The recombinant proteins stably expressed in Escherichia coli cells were purified from culture supernatants in a single step using Ni(2+)-chelating chromatography producing soluble proteins of 34 kDa (LiRecDT4) and 37 kDa (LiRecDT5). Circular dichroism analysis evidenced correctly folding for toxins but differences in secondary structures. Both proteins were recognized by whole venom serum antibodies and by a specific antibody to dermonecrotic toxin. Also, recombinant toxins with phospholipase activity induced experimental skin lesions and caused a massive inflammatory response in rabbit skin dermis. Nevertheless, toxins displayed different effects upon platelet aggregation, increase in vascular permeability and not caused death in mice. These characteristics in combination with functional studies illustrates that a family of dermonecrotic toxins exists, and includes two novel members that are useful for future structural and functional studies. They will also be useful in biotechnological ends, for example, as inflammatory and platelet aggregating studies, as antigens for serum therapy source and for lipids biochemical research.     

Speciation of phytate ion in aqueous solution. Protonation constants and copper(II) interactions in NaNO3aq at different ionic strengths

The acid base behavior of phytate has been studied (at t=25 degrees C by potentiometry, ISE-H+ glass electrode) in NaNO3aq at different ionic strengths (0.1 < or = I/mol L(-1) < or = 1.0). The interactions with copper(II) were investigated in the same experimental conditions in different metal to ligand (Phy) ratios (1:1 < or = Cu2+ :Phy < or = 4:1), by using both ISE-H+ and ISE-Cu2+ electrodes. Phytate acid base behavior in sodium nitrate is very similar to that in sodium chloride, previously investigated. In the experimental conditions adopted, the formation of three CuiHjPhy(12-2i-j)- species is observed: the mononuclear CuH4Phy6- and CuH5Phy5-, and the dinuclear Cu2H5Phy3-. Analysis of complex formation constants at different ionic strengths reveals that both ISE-H+ and ISE-Cu2+ electrodes gave, within the experimental error, analogous values. Dependence of complex formation constants on ionic strength was modeled by EDH (Extended Debye-Hückel) and SIT (Specific ion Interaction Theory) equations. The sequestering ability of phytate toward copper(II) has been evaluated by the calculation of pL50 (the total ligand concentration, as -log CL, able to bind 50% of metal cation), an empirical parameter already proposed for an objective "quantification" of this ability. A thorough analysis of literature data on phytate-copper(II) complexes has been performed.     

Identification of a novel potential antigen for early-phase serodiagnosis of leptospirosis

This study examined four genes encoding for predicted membrane proteins selected from the genome sequences of Leptospira interrogans. Genes were cloned and the proteins expressed in E. coli. Immunoblotting analysis of the recombinants with sera from early and convalescent phases of a leptospirosis patient showed that two proteins, namely Lp29 and Lp49, were reactive with serum from both phases of the illness. These data were further confirmed in enzyme-linked immunosorbent assay using sera from both phases of seventeen confirmed leptospirosis specimens, suggesting that these proteins are presented to the host immune system during infection. In the early phase, anti-Lp29 IgM was detected in all sera when microscopic agglutination tests (MAT), the reference method for diagnosis of leptospirosis, were negative. The gene encoding Lp49 is conserved among five tested leptospiral pathogenic serovars, while Lp29 is present in serovars that are predominant in urban settings. These recombinant antigens might be valuable for serodiagnosis of both phases of leptospirosis.     

Simultaneous monitoring of SARS-CoV-2 and bacterial profiles from the air of hospital environments with COVID-19-affected patients

Aerobiologia - The SARS-CoV-2 presence and the bacterial community profile in air samples collected at the Intensive Care Unit (ICU) of the Operational Unit of Infectious Diseases of Santa Caterina...     

Multisensory mechanisms in temporo-parietal cortex support self-location and first-person perspective

Self-consciousness has mostly been approached by philosophical enquiry and not by empirical neuroscientific study, leading to an overabundance of diverging theories and an absence of data-driven theories. Using robotic technology, we achieved specific bodily conflicts and induced predictable changes in a fundamental aspect of self-consciousness by altering where healthy subjects experienced themselves to be (self-location). Functional magnetic resonance imaging revealed that temporo-parietal junction (TPJ) activity reflected experimental changes in self-location that also depended on the first-person perspective due to visuo-tactile and visuo-vestibular conflicts. Moreover, in a large lesion analysis study of neurological patients with a well-defined state of abnormal self-location, brain damage was also localized at TPJ, providing causal evidence that TPJ encodes self-location. Our findings reveal that multisensory integration at the TPJ reflects one of the most fundamental subjective feelings of humans: the feeling of being an entity localized at a position in space and perceiving the world from this position and perspective.     

β-Gal gene expression MRI reporter in melanoma tumor cells. Design, synthesis, and in vitro and in vivo testing of a Gd(III) containing probe forming a high relaxivity, melanin-like structure upon β-Gal enzymatic activation

The aim of this work is to design and test an MRI probe (Gd-DOTAtyr-gal) able to report on the gene expression of β-galactosidase (β-Gal) in melanoma cells. The probe consists of a Gd-DOTA reporter bearing on its surface a tyrosine-galactose-pyranose functionality that, upon the release of the sugar moiety, readily transforms, in the presence of tyrosinase, into melanin oligomeric/polymeric mixture. The formation of Gd-DOTA-containing melanin oligomers and polymers is accompanied by a marked increase of the water proton relaxation rate. The steps involving the release of the galactose-pyranose group and the formation of the melanin-like structure have been carefully investigated in vitro by relaxometric and UV-vis measurements. Cellular uptake studies of Gd-DOTAtyr-gal by melanoma cells have shown that the probe enters the cells, and it appears not to be confined in endosomal vesicles. Using B16-F10LacZ transfected cells, the fast formation of paramagnetic melanin-Gd(III)-containing species has been assessed by the measurement of increased longitudinal relaxation rates of the cellular pellets suspensions. The in vitro results have been confirmed in in vivo MRI investigations on murine melanoma tumor bearing mice. Upon direct injection of Gd-DOTAtyr-gal, a good contrast is observed after 5 h post injection in B16-F10LacZ tumors, but not in B16-F10 tumors lacking the β-Gal enzyme. Gd-DOTAtyr-gal in combination with tyrosinase introduces a novel approach for the detection of β-Gal expression by MRI in vivo.