Surface properties of glass micropipettes and their effect on biological studies

In this paper, an investigation on surface properties of glass micropipettes and their effect on biological applications is reported. Pipettes were pulled under different pulling conditions and the effect of each pulling parameter was analyzed. SEM stereoscopic technique was used to reveal the surface roughness properties of pipette tip and pipette inner wall in 3D. More than 20 pipettes were reconstructed. Pipette heads were split open using focused ion beam (FIB) milling for access to the inner walls. It is found that surface roughness parameters are strongly related on the tip size. Bigger pipettes have higher average surface roughness and lower developed interfacial area ratio. Furthermore, the autocorrelation of roughness model of the inner surface shows that the inner surface does not have any tendency of orientation and is not affected by pulling direction. To investigate the effect of surface roughness properties on biological applications, patch-clamping tests were carried out by conventional and FIB-polished pipettes. The results of the experiments show that polished pipettes make significantly better seals. The results of this work are of important reference value for achieving pipettes with desired surface properties and can be used to explain biological phenomenon such as giga-seal formation

Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli : purification , biochemical and kinetic characterisation

We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl-aminopeptidase I. The amino acid sequence, deduced from the nucleotide sequence, revealed that it consists of 209 amino acid residues and showed to have 98% homology with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary among the two sequences. The bovine enzyme was expressed in XL10-gold Esherichia coli cells. Immobilizied Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 3.3mg of PAP1 per litre culture. The purified enzyme had a specific activity of 1700 units/ml. SDS-PAGE produced a single band for bovine PAP1 with a molecular weight of ~23-24 kDa which is in good agreement with previously reported data on PAP1. Kinetic constants Km and Kcat were 59μΜ and 3.5s-1, respectively. It possessed an optimum pH between 9-9.5, a temperature of 37°C and showed an absolute requirement for a thiol-reducing agent (10mM DTT). EDTA didn’t prove to have an effect on enzyme activity. Competitive inhibition was seen with pyroglutamyl peptides pGlu-His-Pro-NH2 (TRH; Ki= 44.1 uM), pGlu-Ala- OH (Ki=141 uM) and pGlu-Val-OH (Ki=652.17)

The Ankyrin Repeat Domain of the TRPA Protein Painless Is Important for Thermal Nociception but Not Mechanical Nociception

The Drosophila TRPA channel Painless is required for the function of polymodal nociceptors which detect noxious heat and noxious mechanical stimuli. These functions of Painless are reminiscent of mammalian TRPA channels that have also been implicated in thermal and mechanical nociception. A popular hypothesis to explain the mechanosensory functions of certain TRP channels proposes that a string of ankyrin repeats at the amino termini of these channels acts as an intracellular spring that senses force. Here, we describe the identification of two previously unknown Painless protein isoforms which have fewer ankyrin repeats than the canonical Painless protein. We show that one of these Painless isoforms, that essentially lacks ankyrin repeats, is sufficient to rescue mechanical nociception phenotypes of painless mutant animals but does not rescue thermal nociception phenotypes. In contrast, canonical Painless, which contains Ankyrin repeats, is sufficient to largely rescue thermal nociception but is not capable of rescuing mechanical nociception. Thus, we propose that in the case of Painless, ankryin repeats are important for thermal nociception but not for mechanical nociception

Detection of Neural Activity in the Brains of Japanese Honeybee Workers during the Formation of a “Hot Defensive Bee Ball”

Anti-predator behaviors are essential to survival for most animals. The neural bases of such behaviors, however, remain largely unknown. Although honeybees commonly use their stingers to counterattack predators, the Japanese honeybee (Apis cerana japonica) uses a different strategy to fight against the giant hornet (Vespa mandarinia japonica). Instead of stinging the hornet, Japanese honeybees form a “hot defensive bee ball” by surrounding the hornet en masse, killing it with heat. The European honeybee (A. mellifera ligustica), on the other hand, does not exhibit this behavior, and their colonies are often destroyed by a hornet attack. In the present study, we attempted to analyze the neural basis of this behavior by mapping the active brain regions of Japanese honeybee workers during the formation of a hot defensive bee ball. First, we identified an A. cerana homolog (Acks = Apis cerana kakusei) of kakusei, an immediate early gene that we previously identified from A. mellifera, and showed that Acks has characteristics similar to kakusei and can be used to visualize active brain regions in A. cerana. Using Acks as a neural activity marker, we demonstrated that neural activity in the mushroom bodies, especially in Class II Kenyon cells, one subtype of mushroom body intrinsic neurons, and a restricted area between the dorsal lobes and the optic lobes was increased in the brains of Japanese honeybee workers involved in the formation of a hot defensive bee ball. In addition, workers exposed to 46°C heat also exhibited Acks expression patterns similar to those observed in the brains of workers involved in the formation of a hot defensive bee ball, suggesting that the neural activity observed in the brains of workers involved in the hot defensive bee ball mainly reflects thermal stimuli processing

Microtubule Dynamics Regulate Cyclic Stretch-Induced Cell Alignment in Human Airway Smooth Muscle Cells

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma

Nociceptors: a phylogenetic view

The ability to react to environmental change is crucial for the survival of an organism and an essential prerequisite is the capacity to detect and respond to aversive stimuli. The importance of having an inbuilt “detect and protect” system is illustrated by the fact that most animals have dedicated sensory afferents which respond to noxious stimuli called nociceptors. Should injury occur there is often sensitization, whereby increased nociceptor sensitivity and/or plasticity of nociceptor-related neural circuits acts as a protection mechanism for the afflicted body part. Studying nociception and nociceptors in different model organisms has demonstrated that there are similarities from invertebrates right through to humans. The development of technology to genetically manipulate organisms, especially mice, has led to an understanding of some of the key molecular players in nociceptor function. This review will focus on what is known about nociceptors throughout the Animalia kingdom and what similarities exist across phyla; especially at the molecular level of ion channels