Light- and electron-microscopic analysis of the statocyst of the American crayfishOrconectes limosus (Crustacea,Decapoda)

Summary The statocyst ofOrconectes limosus contains static hairs arranged in four groups. All the hairs are the same in basic structure; they differ only in length and diameter and in their positions with respect to the other hairs in the group and to the statolith. In terms of functional morphology, each static hair is part of a unit consisting of an acellular lever string, three receptor cells, a scolopale cell, sheath cells, and enveloping cells. The lever string comprises two components in a characteristic longitudinal arrangement. The structure of the receptor cells resembles that of the arthropod chemo- and mechanoreceptors studied previously. The cilium and the postciliary section lie within two receptor cavities, formed by the scolopale cell and the sheath cells; the two cavities communicate with one another. The receptor cells are fixed in position by various structures. Proximally they form desmosomes with the scolopale cell, medially they are joined by filaments to the inner wall of cavity 1, and distally they are retained by a constriction between the two cavities. Two possible stimulus-mediating mechanisms are discussed: pressure changes in the receptor cavities and shearing of the base of the cilia with respect to the preciliary region. The lever string is part of the cuticle and hence is shed during molting. Nevertheless, the statocyst remains functional during this process because new structural units are formed below the old cuticle prior to ecdysis.Abbreviations a axon - b bulb - bb basal body - c cilium - cu cuticle - d dendrite - de desmosome - dm dense material - ec enveloping cell - f fulcrum - h hair - hs hair shaft - ir inner row of hair group - l lingula - ls lever string - m mitochondrion - n nucleus - or outer row of hair group - pcd postciliary dilation - R1 receptor cavity 1 - R2 receptor cavity 2 - rc receptor cell - ro rootlet - s sheath cell - sc scolopale cell - st statoconium - t tooth     

The Major Brain Endocannabinoid 2-AG Controls Neuropathic Pain and Mechanical Hyperalgesia in Patients with Neuromyelitis Optica

Recurrent myelitis is one of the predominant characteristics in patients with neuromyelitis optica (NMO). While paresis, visual loss, sensory deficits, and bladder dysfunction are well known symptoms in NMO patients, pain has been recognized only recently as another key symptom of the disease. Although spinal cord inflammation is a defining aspect of neuromyelitis, there is an almost complete lack of data on altered somatosensory function, including pain. Therefore, eleven consecutive patients with NMO were investigated regarding the presence and clinical characteristics of pain. All patients were examined clinically as well as by Quantitative Sensory Testing (QST) following the protocol of the German Research Network on Neuropathic Pain (DFNS). Additionally, plasma endocannabinoid levels and signs of chronic stress and depression were determined. Almost all patients (10/11) suffered from NMO-associated neuropathic pain for the last three months, and 8 out of 11 patients indicated relevant pain at the time of examination. Symptoms of neuropathic pain were reported in the vast majority of patients with NMO. Psychological testing revealed signs of marked depression. Compared to age and gender-matched healthy controls, QST revealed pronounced mechanical and thermal sensory loss, strongly correlated to ongoing pain suggesting the presence of deafferentation-induced neuropathic pain. Thermal hyperalgesia correlated to MRI-verified signs of spinal cord lesion. Heat hyperalgesia was highly correlated to the time since last relapse of NMO. Patients with NMO exhibited significant mechanical and thermal dysesthesia, namely dynamic mechanical allodynia and paradoxical heat sensation. Moreover, they presented frequently with either abnormal mechanical hypoalgesia or hyperalgesia, which depended significantly on plasma levels of the endogenous cannabinoid 2-arachidonoylglycerole (2-AG). These data emphasize the high prevalence of neuropathic pain and hyperalgesia in patients with NMO. The degree of mechanical hyperalgesia reflecting central sensitization of nociceptive pathways seems to be controlled by the major brain endocannabinoid 2-AG.     

Improving the autotransporter-based surface display of enzymes in Pseudomonas putida KT2440

Pseudomonas putida can be used as a host for the autotransporter-mediated surface display of enzymes (autodisplay), resulting in whole-cell biocatalysts with recombinant functionalities on their cell envelope. The efficiency of autotransporter-mediated secretion depends on the N-terminal signal peptide as well as on the C-terminal translocator domain of autotransporter fusion proteins. We set out to optimize autodisplay for P. putida as the host bacterium by comparing different signal peptides and translocator domains for the surface display of an esterase. The translocator domain did not have a considerable effect on the activity of the whole-cell catalysts. In contrast, by using the signal peptide of the P. putida outer membrane protein OprF, the activity was more than 12-fold enhanced to 638 mU ml⁻¹ OD⁻¹ compared with the signal peptide of V. cholerae CtxB (52 mU ml⁻¹ OD⁻¹). This positive effect was confirmed with a β-glucosidase as a second example enzyme. Here, cells expressing the protein with N-terminal OprF signal peptide showed more than fourfold higher β-glucosidase activity (181 mU ml⁻¹ OD⁻¹) than with the CtxB signal peptide (42 mU ml⁻¹ OD⁻¹). SDS-PAGE and flow cytometry analyses indicated that the increased activities correlated with an increased amount of recombinant protein in the outer membrane and a higher number of enzymes detectable on the cell surface.     

Agonists of Orally Expressed TRP Channels Stimulate Salivary Secretion and Modify the Salivary Proteome

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Highlights

• •Salivary secretion was increased by mouth rinsing with TRP channel agonists.
• •The salivary proteome varied over time and was changed by TRP channel stimulation.
• •Immunoreactive Cystatin S was increased in saliva after TRPV1 stimulation.

     

Quantifying Protein-Protein Interactions of Peripheral Membrane Proteins by Fluorescence Brightness Analysis

Fluorescently labeled proteins that are found both in the cytoplasm and at the plasma membrane, such as peripheral membrane proteins, create stratified fluorescent layers that present a challenging environment for brightness studies with fluorescence fluctuation spectroscopy. The geometry of each layer along with fluorescence and brightness contributions from adjacent layers generates a convoluted raw brightness that conceals the underlying brightness of each individual layer. Because the brightness at a layer establishes the oligomeric state of the fluorescently labeled protein at said layer, we developed a method that connects the experimental raw brightness with the physical brightness at each layered compartment. The technique determines the oligomerization in each compartment from an axial intensity scan through the sample, followed by a fluorescence fluctuation spectroscopy measurement at each layer. We experimentally verify the technique with H-Ras-EGFP as a model system and determine its oligomeric state at both the plasma membrane and in the cytoplasm. Furthermore, we study the oligomerization of the Gag matrix domain of Human T-lymphotropic virus Type 1. The matrix domain targets the Gag polyprotein to the plasma membrane where, subsequently, viral assembly occurs. We determine the oligomerization of matrix in the cytoplasm and observe the onset of protein-protein interactions at the membrane. These observations shed light on the early assembly steps of the retrovirus.