Synaptobrevin/vesicle-associated membrane protein (VAMP) of Aplysia californica: structure and proteolysis by tetanus toxin and botulinal neurotoxins type D and F.

Synaptobrevin/vesicle-associated membrane protein (VAMP) and syntaxin are potential vesicle donor and target membrane receptors of a docking complex that requires N-ethylmaleimide-sensitive factor (NSF) and soluble NSF-attachment proteins as soluble factors for vesicle fusion with target membranes. Members of this docking complex are the target of clostridial neurotoxins that act as zinc-dependent proteases. Molecular cloning of the Aplysia californica synaptobrevin cDNA revealed a 180-residue polypeptide (M(r), 19,745) with a central transmembrane region and an atypically large C-terminal intravesicular domain. This polypeptide integrates into membranes at both the co- and posttranslational level, as shown by modification of an artificially introduced N-glycosylation site. The soluble and membrane-anchored forms of synaptobrevin are cleaved by the light chains of the botulinal toxins type D and F and by tetanus toxin involving the peptide bonds Lys49-Ile50, Gln48-Lys49, and Gln66-Phe67, respectively. The active center of teh tetanus toxin light chain was identified by site-specific mutagenesis. His233, His237, Glu234, and Glu270/271 are essential to this proteolytic activity. Modification of histidine residues resulted in loss of zinc binding, whereas a replacement of Glu234 only slightly reduced the zinc content.     

Variable cooperativity in SNARE-mediated membrane fusion

The soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex drives the majority of intracellular and exocytic membrane fusion events. Whether and how SNAREs cooperate to mediate fusion has been a subject of intense study, with estimates ranging from a single SNARE complex to 15. Here we show that there is no universally conserved number of SNARE complexes involved as revealed by our observation that this varies greatly depending on membrane curvature. When docking rates of small (∼40 nm) and large (∼100 nm) liposomes reconstituted with different synaptobrevin (the SNARE present in synaptic vesicles) densities are taken into account, the lipid mixing efficiency was maximal with small liposomes with only one synaptobrevin, whereas 23–30 synaptobrevins were necessary for efficient lipid mixing in large liposomes. Our results can be rationalized in terms of strong and weak cooperative coupling of SNARE complex assembly where each mode implicates different intermediate states of fusion that have been recently identified by electron microscopy. We predict that even higher variability in cooperativity is present in different physiological scenarios of fusion, and we further hypothesize that plasticity of SNAREs to engage in different coupling modes is an important feature of the biologically ubiquitous SNARE-mediated fusion reactions.Membrane fusion is an essential reaction common to intracellular trafficking and exocytosis in eukaryotic cells. Although the process involves an intricate interplay of several proteins, the fusion of membranes is dependent on the conserved family of proteins known as soluble N-ethylmaleimide–sensitive factor attachment protein receptors, or SNAREs (1, 2). In the important case of the fusion of synaptic vesicles (SVs), the SNAREs responsible are vesicular synaptobrevin 2 (syb) and plasma membrane proteins SNAP-25A (SN25) and syntaxin-1A (syx). A critical intermediate seems to be an acceptor complex consisting of a three-helix bundle formed by a 1:1 syx:SN25 complex, which serves as a binding site for syb (3, 4). According to the zipper hypothesis, the N termini of syb and the 1:1 syx:SN25 complex nucleate to form a parallel four-helix bundle called the SNARE complex. The directional assembly then proceeds toward the C termini, resulting in a pulling force between the membranes that leads to their fusion (4, 5). There is some consensus that the highly exergonic nature of the assembly of the SNARE complex provides the energy for overcoming the barrier for fusion (6, 7), although identification of putative fusion intermediates at molecular resolution as well as force measurement experiments suggest multiple energy barriers are present (7–10).The question of whether and how SNAREs cooperate to mediate fusion has received substantial attention. Although some studies have left open the possibility that the number of SNARE complexes that cooperate during fusion is variable (11, 12), much attention has been given to the notion of a preferred number of SNARE complexes, with estimates varying from a single SNARE complex (13) to 15 (14), although more recent estimates vary between two and eight (12, 15–18). Unfortunately, this large disparity in results has not been appropriately explained, and it remains unclear whether the differences are a result of inherent properties of the particular set of SNAREs involved or rather originate from the biophysical characteristics of the fusing vesicles.A commonly used approach to investigate how SNAREs work is by reconstituting complementary SNAREs into liposomes (19). We have previously demonstrated that SNAREs can mediate both lipid and contents mixing with similar kinetics (13), an important functional criterion for establishing membrane fusion (20). Using the SNAREs responsible for exocytosis of SVs as a model, we investigate here how the density of SNAREs affects fusion of liposomes to obtain mechanistic information on cooperativity. A rigorous way to address this is to vary the SNARE density in one or both membranes. Whereas it is experimentally straightforward for syb, this approach is problematic for SN25 and syx because they tend to associate into “off-pathway” complexes, compromising kinetic analysis (6, 21). To circumvent this, Pobbati et al. (22) introduced a stabilized acceptor complex that contains a C-terminal peptide of synaptobrevin (syb 49–96) (22). This 1:1 syx:SN25 complex (herein referred to as the ∆N complex) is stable and contains a free binding site for syb, allowing one to precisely define its concentration. Therefore, we used the ∆N complex in our experiments to rule out the effect of any side reactions related to the assembly/disassembly of the acceptor complex and thus simplify the kinetic analysis on fusion.     

Gait characteristics of patients with phobic postural vertigo: effects of fear of falling,attention, and visual input

Phobic postural vertigo (PPV) is the most common cause of chronic dizziness in middle-aged patients. Many patients report symptoms involving gait. We investigated the gait performance and its relationship to the fear of falling and attention of PPV patients in a prospective study of 24 patients with PPV and 24 healthy subjects (HS) using a pressure-sensitive mat (GAITRite^®). Subjects walked at three different speeds (slow, preferred, fast), both during cognitive dual tasks (DTc) and with eyes closed (EC). Falls efficacy and balance confidence were rated by the Falls Efficacy Scale-International (FES-I) and the Activities-specific Balance Confidence Scale (ABC). PPV patients walked slower, with reduced cadence (all p < 0.01), stride length (p < 0.05), and increased double support (p < 0.01) compared to HS. These changes correlated with FES-I (R = ?0.528, p < 0.001) and ABC (R = 0.481, p < 0.01). Walking deterioration under DTc did not differ between PPV patients and HS, but patients showed a reduced cognitive processing speed (p < 0.05). When walking with EC, gait speed decreased more in PPV patients compared to HS (p < 0.05). Patients with PPV show gait changes which correlate with their fear of falling and balance confidence. Absent visual feedback leads to more pronounced gait deteriorations in PPV patients than in HS, indicating a higher reliance of patients on visual information during walking. These findings support the view that the gait characteristics of PPV can be attributed to an inadequate, cautious gait control.     

Hyalinizing trabecular adenoma: A misnomer for a peculiar tumor of the thyroid gland

We describe the clinical, histological, immunohistochemical, and electron microscopical features of 9 tumors fulfilling the criteria of the so-called hyalinizing trabecular adenoma (HTA) of the thyroid. Six tumors had the characteristic histology of HTA throughout, whereas in the remaining 3 tumors the classic pattern was identified focally in otherwise typical or atypical follicular adenomas. In one case, there was a focus of tumoral tissue outside the capsule, and in another there was a regional lymph node metastasis. Seven tumors were immunoreactive for thyroglobulin and cytokeratins, 1 tumor was positive for thyroglobulin and negative for cytokeratins, and another was negative for thyroglobulin and positive for cytokeratins. Scattered cells immunoreactive for neurotensin and somatostatin were found in 2 cases. Every tumor stained for S100 protein and neuron-specific enolase, but none showed immunoreactivity for calcitonin, calcitonin gene-related peptide, or chromogranin. The irregularity of the nuclear contours, the prominence of the cytoplasmic bundles of intermediate filaments, and the accumulation of basal lamina material around the neoplastic cells without the interposition of a well-defined basal lamina were the most distinctive electron microscopical features. The cytogenetic study performed in one case revealed, apart from cells with a normal karyotype, two abnormal clones: one with a translocation of chromosomes 2-3 and another with the same translocation and trisomies of chromosomes 7 and 12. Our results show that most HTAs display follicular cell differentiation and very low clinical aggressiveness. They also show that some HTAs are able to coexpress follicular cell and neuroendocrine markers and may behave like malignant neoplasms. We conclude that hyalinizing trabecular tumor is a more appropriate generic term than HTA to designate this relatively heterogenous group of lesions of the thyroid gland.     

Patients with chronic pain exhibit individually unique cortical signatures of pain encoding

Chronic pain is characterised by an ongoing and fluctuating intensity over time. Here, we investigated how the trajectory of the patients'' endogenous pain is encoded in the brain. In repeated functional MRI (fMRI) sessions, 20 patients with chronic back pain and 20 patients with chronic migraine were asked to continuously rate the intensity of their endogenous pain. Linear mixed effects models were used to disentangle cortical processes related to pain intensity and to pain intensity changes. At group level, we found that the intensity of pain in patients with chronic back pain is encoded in the anterior insular cortex, the frontal operculum, and the pons; the change of pain in chronic back pain and chronic migraine patients is mainly encoded in the anterior insular cortex. At the individual level, we identified a more complex picture where each patient exhibited their own signature of endogenous pain encoding. The diversity of the individual cortical signatures of chronic pain encoding results bridge between clinical observations and neuroimaging; they add to the understanding of chronic pain as a complex and multifaceted disease.     

Effects of 2,4-dichlorophenoxyacetic acid on the ventral prostate of rats during the peri-pubertal,pubertal and adult stage

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is used on a wide variety of terrestrial and aquatic broadleaf weeds. 2,4-D has been shown to produce a wide range of adverse effects on animal and human health. The aim of the current study was to evaluate the effects of pre- and postnatal exposure to 2,4-D on rat ventral prostate (VP). Pregnant rats were exposed daily to oral doses of 70?mg/kg/day of 2,4-D from 16 days of gestation up to 23 days after delivery. Then, the treated groups (n?=?8) were fed with a 2,4-D added diet until sacrificed by decapitation on postnatal day (PND) 45, 60, or 90. Morphometric studies were performed and androgen receptor (AR) protein levels in the VP were determined. AR, insulin-like growth factor-I (IGF-1) and insulin-like growth factor-I receptor (IGF-1R) mRNA expression in the VP along with testosterone (T), dihydroxytestosterone (DHT), growth hormone (GH) and IGF-1 serum levels were also determined to ascertain whether these parameters were differentially affected. Results of this study showed that 2,4-D exposure during gestation and until adulthood altered development of the prostate gland in male rats, delaying it at early ages while increasing its size in adults, indicate that 2,4-D could behave as endocrine disruptors (EDs).