Up-regulation of Cavβ3 subunit in primary sensory neurons increases voltage-activated Ca2+ channel activity and nociceptive input in neuropathic pain

High voltage-activated calcium channels (HVACCs) are essential for synaptic and nociceptive transmission. Although blocking HVACCs can effectively reduce pain, this treatment strategy is associated with intolerable adverse effects. Neuronal HVACCs are typically composed of α(1), β (Cavβ), and α(2)δ subunits. The Cavβ subunit plays a crucial role in the membrane expression and gating properties of the pore-forming α(1) subunit. However, little is known about how nerve injury affects the expression and function of Cavβ subunits in primary sensory neurons. In this study, we found that Cavβ(3) and Cavβ(4) are the most prominent subtypes expressed in the rat dorsal root ganglion (DRG) and dorsal spinal cord. Spinal nerve ligation (SNL) in rats significantly increased mRNA and protein levels of the Cavβ(3), but not Cavβ(4), subunit in the DRG. SNL also significantly increased HVACC currents in small DRG neurons and monosynaptic excitatory postsynaptic currents of spinal dorsal horn neurons evoked from the dorsal root. Intrathecal injection of Cavβ(3)-specific siRNA significantly reduced HVACC currents in small DRG neurons and the amplitude of monosynaptic excitatory postsynaptic currents of dorsal horn neurons in SNL rats. Furthermore, intrathecal treatment with Cavβ(3)-specific siRNA normalized mechanical hyperalgesia and tactile allodynia caused by SNL but had no significant effect on the normal nociceptive threshold. Our findings provide novel evidence that increased expression of the Cavβ(3) subunit augments HVACC activity in primary sensory neurons and nociceptive input to dorsal horn neurons in neuropathic pain. Targeting the Cavβ(3) subunit at the spinal level represents an effective strategy for treating neuropathic pain.     

Identification of diagnostic biomarkers for infection in premature neonates

Infection is a leading cause of neonatal morbidity and mortality worldwide. Premature neonates are particularly susceptible to infection because of physiologic immaturity, comorbidity, and extraneous medical interventions. Additionally premature infants are at higher risk of progression to sepsis or severe sepsis, adverse outcomes, and antimicrobial toxicity. Currently initial diagnosis is based upon clinical suspicion accompanied by nonspecific clinical signs and is confirmed upon positive microbiologic culture results several days after institution of empiric therapy. There exists a significant need for rapid, objective, in vitro tests for diagnosis of infection in neonates who are experiencing clinical instability. We used immunoassays multiplexed on microarrays to identify differentially expressed serum proteins in clinically infected and non-infected neonates. Immunoassay arrays were effective for measurement of more than 100 cytokines in small volumes of serum available from neonates. Our analyses revealed significant alterations in levels of eight serum proteins in infected neonates that are associated with inflammation, coagulation, and fibrinolysis. Specifically P- and E-selectins, interleukin 2 soluble receptor alpha, interleukin 18, neutrophil elastase, urokinase plasminogen activator and its cognate receptor, and C-reactive protein were observed at statistically significant increased levels. Multivariate classifiers based on combinations of serum analytes exhibited better diagnostic specificity and sensitivity than single analytes. Multiplexed immunoassays of serum cytokines may have clinical utility as an adjunct for rapid diagnosis of infection and differentiation of etiologic agent in neonates with clinical decompensation.     

Comparative study on the gametophyte morphology and development of three paramo species of Jamesonia (Pteridaceae,Polypodiopsida)

The sexual phase of three species of Jamesonia (J. imbricata, J. rotundifolia and J. scammaniae) has been studied, from spore germination to gametangia formation, with special attention to the morphological development of prothalli. This is the first work to deal with gametophytes of species in this genus. All three species have trilete, tetrahedrical spores that germinate following the Vittaria type. The developmental pattern of J. imbricata and J. rotundifolia is intermediate between the Adiantum and Ceratopteris types, as the initial mitotic activity is assumed by an obconical apical cell, substituted later by a lateral meristem. In J. scammaniae, no apical cell or lateral meristem is formed, so the prothalli remain ameristic throughout the developmental process. Adult gametophytes are naked and variable in shape, in the same species ranging from typical cordate prothalli to irregularly lobed forms. Gametangia of normal shape and size was observed in both J. imbricata and J. rotundifolia, but not in J. scammaniae. Apogamy could be expected to occur in the genus, a phenomenon to be examined in the future. Comparisons are made with known species of related genera and results are discussed from an ecological perspective in the paramo environment.     

Potentiation of direct antitumor cytotoxicity and production of tumor cytolytic factors in human blood monocytes by human recombinant interferon-gamma and muramyl dipeptide derivatives

Summary We investigated whether human peripheral blood monocytes isolated by centrifugal elutriation from healthy donors could be acitivated to become tumoricidal and release tumor cytolytic factor (TCF) subsequent to incubation with recombinant human interferon-gamma (r-IFN-) or a derivative of muramyl dipeptide (nor-MDP), or both. Blood monocytes incubated in endotoxin-free medium containing up to 1000 U/ml of r-IFN- or in medium containing less than 1 g/ml of nor MDP were not activated to lyse radiolabeled allogeneic human tumor cells. In contrast, the incubation of monocytes with various dose combinations of r-IFN- and nor-MDP generated significant direct cytotoxic activity as well as production of TCF. Preincubation of the r-IFN- and nor-MDP mixture with polymyxin B did not inhibit the synergism, thus ruling out the possibility that the process was due to endotoxin contamination. TCF harvested from monocyte culture supernatants was cytolytic against five allogeneic tumor targets, but not against a nontumorigenic cell line. Collectively, the data demonstrate that r-IFN- can prime human blood monocytes to allow their activation by synthetic nor-MDP.On leave from the Department of Internal Medicine, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima 770, Japan     

Graph Based Study of Allergen Cross-Reactivity of Plant Lipid Transfer Proteins (LTPs) Using Microarray in a Multicenter Study

The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.     

Intronic Variants in the NFKB1 Gene May Influence Hearing Forecast in Patients with Unilateral Sensorineural Hearing Loss in Meniere's Disease

Meniere''s disease is an episodic vestibular syndrome associated with sensorineural hearing loss (SNHL) and tinnitus. Patients with MD have an elevated prevalence of several autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis and psoriasis), which suggests a shared autoimmune background. Functional variants of several genes involved in the NF-κB pathway, such as REL, TNFAIP3, NFKB1 and TNIP1, have been associated with two or more immune-mediated diseases and allelic variations in the TLR10 gene may influence bilateral affectation and clinical course in MD. We have genotyped 716 cases of MD and 1628 controls by using the ImmunoChip, a high-density genotyping array containing 186 autoimmune loci, to explore the association of immune system related-loci with sporadic MD. Although no single nucleotide polymorphism (SNP) reached a genome-wide significant association (p<10⁻⁸), we selected allelic variants in the NF-kB pathway for further analyses to evaluate the impact of these SNPs in the clinical outcome of MD in our cohort. None of the selected SNPs increased susceptibility for MD in patients with uni or bilateral SNHL. However, two potential regulatory variants in the NFKB1 gene (rs3774937 and rs4648011) were associated with a faster hearing loss progression in patients with unilateral SNHL. So, individuals with unilateral MD carrying the C allele in rs3774937 or G allele in rs4648011 had a shorter mean time to reach hearing stage 3 (>40 dB HL) (log-rank test, corrected p values were p = 0.009 for rs3774937 and p = 0.003 for rs4648011, respectively). No variants influenced hearing in bilateral MD. Our data support that the allelic variants rs3774937 and rs4648011 can modify hearing outcome in patients with MD and unilateral SNHL.     

Thermoluminescence and P700 redox kinetics as complementary tools to investigate the cyclic/chlororespiratory electron pathways in stress conditions in barley leaves

Cyclic electron flow around photosystem I drives additional proton pumping into the thylakoid lumen, which enhances the protective non-photochemical quenching and increases ATP synthesis. It involves several pathways activated independently. In whole barley leaves, P700 oxidation under far-red illumination and subsequent P700(+) dark reduction kinetics provide a major probe of the activation of cyclic pathways. Two 'intermediate' and 'slow' exponential reduction phases are always observed and they become faster after high light illumination, but dark inactivation of the Benson-Calvin cycle causes the emergence of both a transient in the P700 oxidation and a 'fast' phase in the P700(+) reduction. We investigate here the afterglow (AG) thermoluminescence emission as another tool to detect the activation of cyclic electron pathways from stroma reductants to the acceptor side of photosystem II. This transfer is activated by warming, yielding an AG band at about 45°C. However, treatments that accelerate the 'intermediate' and 'slow' P700(+) reduction phases (brief anoxia, hexose infiltration, fast dehydration of excised leaves) also produced a downshift of this AG band. This pathway ascribable to NADPH dehydrogenase (NDH) would be triggered by a deficit in ATP, while the 'fast' reduction phase corresponding to the ferredoxin plastoquinone reductase pathway is triggered by an overreduction of the photosystem I acceptor pool and is undetected in thermoluminescence. Contrastingly, slow dehydration of unwatered plants did not cause faster reduction of P700(+) nor temperature downshift of the AG band, that is no induction of the NDH pathway, whereas an increased intensity of the AG band indicated a strong NADPH + ATP assimilatory potential.     

The wobble nucleotide-excising anticodon nuclease RloC is governed by the zinc-hook and DNA-dependent ATPase of its Rad50-like region

The conserved bacterial anticodon nuclease (ACNase) RloC and its phage-excluding homolog PrrC comprise respective ABC-adenosine triphosphatase (ATPase) and ACNase N- and C-domains but differ in three key attributes. First, prrC is always linked to an ACNase silencing, DNA restriction-modification (R-M) locus while rloC rarely features such linkage. Second, RloC excises its substrate's wobble nucleotide, a lesion expected to impede damage reversal by phage transfer RNA (tRNA) repair enzymes that counteract the nick inflicted by PrrC. Third, a distinct coiled-coil/zinc-hook (CC/ZH) insert likens RloC's N-region to the universal DNA damage checkpoint/repair protein Rad50. Previous work revealed that ZH mutations activate RloC's ACNase. Data shown here suggest that RloC has an internal ACNase silencing/activating switch comprising its ZH and DNA-break-responsive ATPase. The existence of this control may explain the lateral transfer of rloC without an external silencer and supports the proposed role of RloC as an antiviral contingency acting when DNA restriction is alleviated under genotoxic stress. We also discuss RloC's possible evolution from a PrrC-like ancestor.