Toxins2015, 7(7), 2534-2550; doi:10.3390/toxins7072534 (registering DOI) - published 6 July 2015 Show/Hide Abstract
Abstract: The toxin, previously described as a “non-toxic” toxin, was isolated from the scorpion venom of Tityus serrulatus (Ts), responsible for the most severe and the highest number of accidents in Brazil. In this study, the subtype specificity and selectivity of Ts4 was investigated using six mammalian Nav channels (Nav1.2→Nav1.6 and Nav1.8) and two insect Nav channels (DmNav1 and BgNav). The electrophysiological assays showed that Ts4 specifically inhibited the fast inactivation of Nav1.6 channels, the most abundant sodium channel expressed in the adult central nervous system, and can no longer be classified as a “non-toxic peptide”. Based on the results, we could classify the Ts4 as a classical α-toxin. The Ts4 3D-structural model was built based on the solved X-ray Ts1 3D-structure, the major toxin from Ts venom with which it shares high sequence identity (65.57%). The Ts4 model revealed a flattened triangular shape constituted by three-stranded antiparallel β-sheet and one α-helix stabilized by four disulfide bonds. The absence of a Lys in the first amino acid residue of the N-terminal of Ts4 is probably the main responsible for its low toxicity. Other key amino acid residues important to the toxicity of α- and β-toxins are discussed here.
Abstract: Available genomic data for the toxic, bloom-forming, benthic Ostreopsis spp. are traditionally obtained from isolates rather than from individuals originally present in environmental samples. Samples from the final phase of the first reported Ostreopsis bloom in European North Atlantic waters (Algarve, south coast of Portugal) were studied and characterized, using a culture-independent approach. In the first instance, a microscopy-based analysis revealed the intricate complexity of the samples. Then, we evaluated the adequacy of commonly used molecular tools (i.e., primers and nuclear ribosomal markers) for the study of Ostreopsis diversity in natural samples. A PCR-based methodology previously developed to identify/detect common Ostreopsis species was tested, including one new combination of existing PCR primers. Two sets of environmental rRNA sequences were obtained, one of them (1052 bp) with the newly tested primer set. These latter sequences encompass both the ITS1-5.8S-ITS2 region and the D1/D2 domain of the LSU rRNA gene, leading us to an accurate identification of ITS2. In turn, this allowed us to predict and show for the first time the ITS2 secondary structure of Ostreopsis. With 92 bp in length and a two-helix structure, the ITS2 of this genus revealed to be unique among the dinoflagellates. Both the PCR approach as the phylogenetic analyses allowed to place the Ostreopsis cells observed in the samples within the O. cf. ovata phylospecies’ complex, discarding the presence of O. cf. siamensis. The (phylo)genetic results point out a certain level of nucleotide sequence divergence, but were inconclusive in relation to a possible geographic origin of the O. cf. ovata population from the Algarve’s bloom.
Abstract: Voltage-gated sodium (NaV) channels are responsible for propagating action potentials in excitable cells. NaV1.7 plays a crucial role in the human pain signalling pathway and it is an important therapeutic target for treatment of chronic pain. Numerous spider venom peptides have been shown to modulate the activity of NaV channels and these peptides represent a rich source of research tools and therapeutic lead molecules. The aim of this study was to determine the diversity of NaV1.7-active peptides in the venom of an Australian Phlogius sp. tarantula and to characterise their potency and subtype selectivity. We isolated three novel peptides, μ-TRTX-Phlo1a, -Phlo1b and -Phlo2a, that inhibit human NaV1.7 (hNaV1.7). Phlo1a and Phlo1b are 35-residue peptides that differ by one amino acid and belong in NaSpTx family 2. The partial sequence of Phlo2a revealed extensive similarity with ProTx-II from NaSpTx family 3. Phlo1a and Phlo1b inhibit hNaV1.7 with IC50 values of 459 and 360 nM, respectively, with only minor inhibitory activity on rat NaV1.2 and hNaV1.5. Although similarly potent at hNaV1.7 (IC50 333 nM), Phlo2a was less selective, as it also potently inhibited rNaV1.2 and hNaV1.5. All three peptides cause a depolarising shift in the voltage-dependence of hNaV1.7 activation.
Abstract: The aim of this prospective open-label study was to treat disabling drooling in children with cerebral palsy (CP) with onabotulinumtoxin A (A/Ona, Botox®) into submandibular and parotid glands and find the lowest effective dosage and least invasive method. A/Ona was injected in 14 children, Mean age 9 years, SD 3 years, under ultrasonic guidance in six successive Series, with at least six months between injections. Doses and gland involvement increased from Series A to F (units (U) per submandibular/parotid gland: A, 10/0; B, 15/0; C, 20/0; D, 20/20; E, 30/20; and F, 30/30). The effect was assessed 2, 4, 8, 12, and 20 weeks after A/Ona (drooling problems (VAS), impact (0–7), treatment effect (0–5), unstimulated whole saliva (UWS) flow and composition)) and analyzed by two-way ANOVA. The effect was unchanged–moderate in A to moderate–marked in F. Changes in all parameters were significant in E and F, but with swallowing problems ≤5 weeks in 3 of 28 treatments. F had largest VAS and UWS reduction (64% and 49%). We recommend: Start with dose D A/Ona (both submandibular and parotid glands and a total of 80 U) and increase to E and eventually F (total 120 U) without sufficient response.
Abstract: Pain is a natural protective mechanism and has a warning function signaling imminent or actual tissue damage. Neuropathic pain (NP) results from a dysfunction and derangement in the transmission and signal processing along the nervous system and it is a recognized disease in itself. The prevalence of NP is estimated to be between 6.9% and 10% in the general population. This condition can complicate the recovery from stroke, multiple sclerosis, spinal cord lesions, and several neuropathies promoting persistent disability and poor quality of life. Subjects suffering from NP describe it as burning, itching, lancing, and numbness, but hyperalgesia and allodynia represent the most bothersome symptoms. The management of NP is a clinical challenge and several non-pharmacological and pharmacological interventions have been proposed with variable benefits. Botulinum toxin (BTX) as an adjunct to other interventions can be a useful therapeutic tool for the treatment of disabled people. Although BTX-A is predominantly used to reduce spasticity in a neuro-rehabilitation setting, it has been used in several painful conditions including disorders characterized by NP. The underlying pharmacological mechanisms that operate in reducing pain are still unclear and include blocking nociceptor transduction, the reduction of neurogenic inflammation by inhibiting neural substances and neurotransmitters, and the prevention of peripheral and central sensitization. Some neurological disorders requiring rehabilitative intervention can show neuropathic pain resistant to common analgesic treatment. This paper addresses the effect of BTX-A in treating NP that complicates frequent disorders of the central and peripheral nervous system such as spinal cord injury, post-stroke shoulder pain, and painful diabetic neuropathy, which are commonly managed in a rehabilitation setting. Furthermore, BTX-A has an effect in relief pain that may characterize less common neurological disorders including post-traumatic neuralgia, phantom limb, and complex regional pain syndrome with focal dystonia. The use of BTX-A could represent a novel therapeutic strategy in caring for neuropathic pain whenever common pharmacological tools have been ineffective. However, large and well-designed clinical trials are needed to recommend BTX-A use in the relief of neuropathic pain.
Abstract: Botulinum neurotoxins (BoNTs) have been widely used to treat a variety of clinical ailments associated with pain. The inhibitory action of BoNTs on synaptic vesicle fusion blocks the releases of various pain-modulating neurotransmitters, including glutamate, substance P (SP), and calcitonin gene-related peptide (CGRP), as well as the addition of pain-sensing transmembrane receptors such as transient receptor potential (TRP) to neuronal plasma membrane. In addition, growing evidence suggests that the analgesic and anti-inflammatory effects of BoNTs are mediated through various molecular pathways. Recent studies have revealed that the detailed structural bases of BoNTs interact with their cellular receptors and SNAREs. In this review, we discuss the molecular and cellular mechanisms related to the efficacy of BoNTs in alleviating human pain and insights on engineering the toxins to extend therapeutic interventions related to nociception.