Effects of reversible nare occlusion on the development of the olfactory epithelium in the rabbit nasal septum

Summary To investigate environmental influences on the development of the olfactory epithelium, semi-thin sections were taken from the nasal septum of newborn and 30-dayold rabbits; the epithelial thickness and the number of olfactory knobs, supporting cells, dark basal cells, and receptor cells were compared. During normal development, a marked increase in epithelial thickness was found, largely because of an increase in the number of receptor cells. Whereas unilateral nare occlusion on day 1 resulted in 10% fewer receptor cells and 25% fewer knobs on the deprived side by day 30, nare occlusion either up to or after day 5 had little effect, and even temporary reopening from days 6–7 was sufficient to stimulate receptor-cell development on the occluded side. Although in these latter cases, a slight deprivation effect of 6% was still found in the number of receptor-cell nuclei, there was no longer a significant difference in the number of knobs between the open and closed sides. Thus, whereas exposure to the environment during the first days of life appears to be sufficient to stimulate sustained growth, the deprived epithelium may retain the capacity to respond to such cues beyond this time. However, as nare occlusion also had an effect on the respiratory epithelium and nasal lymphatic tissue, the nature of the cues stimulating receptor-cell development, whether olfactory or non-olfactory, is not yet clear.     

On the interaction of cobra venom protein cardiotoxins with erythrocytes

The principally active hemolytic toxin (cardiotoxin) previously purified from the venom of the Thailand cobra, Naja naja siamensis, was shown to produce spontaneous twitching, contractures and membrane depolarization in sartorius muscles from the frog, Rana pipiens. Spontaneous twitching, observed at concentrations greater than 0.1 uM was completely abolished by addition of tetrodotoxin and not affected by d-tubocurarine. Dose and time dependent membrane depolarization of muscle fibers was observed to occur within 10-30 min at 0.2 to 1.0 uM concentrations of the toxin. These observations, taken together with an amino acid analysis characteristic of previously described cobra venom cardiotoxins, characterized this hemolytic toxin as a cardiotoxin. In the absence of EDTA the initial velocities of erythrocyte hemolysis for this toxin showed a sigmoidal concentration dependence which became hyperbolic in the presence of EDTA. The largest increases in hemolysis rates on addition of 1 mM EDTA were observed at low toxin concentrations. In the presence of EDTA extracellular and membrane associated divalent cations are complexed, thus alleviating their competition with toxin for binding to the membrane, a key and apparently rate-determining initial step which leads to hemolysis. In the presence of EDTA hemolysis rates increased linearly at low toxin concentration and reached an extrapolated maximum value at toxin concentrations at which, given its molecular dimensions, there are just sufficient toxin molecules to cover the entire membrane surface area provided by the erythrocytes.     

Principles of separating micro-organisms from suspensions using ultrasound

C.A. MILES, M.J. MORLEY, W.R. HUDSON AND B.M. MACKEY. 1995. When particles in suspension are placed in a stationary ultrasonic field, they form bands of high concentration at half-wavelength intervals. Several theoretical papers have recently contributed to an understanding of the process but, for particles as small as bacteria, there are substantial discrepancies between the different theories, and a lack of published observations.
The threshold amplitudes required to band latex spheres (0.5-5.0 μm diameter) were measured in the frequency range 1–3 MHz and used to establish conditions, reported here for the first time, suitable for banding and moving vegetative bacterial cells in pure culture. Ultrasonic means for separating and concentrating cream and bacteria at opposite ends of a tube containing a mixture of Escherichia coli and diluted milk are also described and the breakdown of theoretical equations at low particle size is discussed.     

Ethidium analogues with improved resolution in the dye-buoyant density procedure.

Analogues of ethidium chloride with large quaternary side chains have been synthesized and evaluated for improved resolution in the dye-buoyant density system for the separation of open and closed circular DNA. These compounds are similar to propidium which differs from ethidium by the replacement of an ethyl group by a methyldiethylaminopropyl group. The new analogues contain a triethylamino group attached to a methylene chain with 3, 5 or 7 carbons. With PM2 DNA the observed separation for propidium is 1.97 times that of ethidium and the new dyes show separations relative to ethidium of 2.27, 2.66 and 2.77. A correlation is established between the mass of the dye component and the observed separation which is retionalized on the basis of the four component thermodynamics describing this system.     

Concomitant Action of Structural Elements and Receptor Phosphorylation Determines Arrestin-3 Interaction with the Free Fatty Acid Receptor FFA4

In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implicated in the regulation of metabolic and inflammatory responses. Here we show, using mass spectrometry, mutagenesis, and phosphospecific antibodies, that agonist-regulated phosphorylation of the human FFA4 receptor occurred primarily at five residues (Thr³⁴⁷, Thr³⁴⁹, Ser³⁵⁰, Ser³⁵⁷, and Ser³⁶⁰) in the C-terminal tail. Mutation of these residues reduced both the efficacy and potency of ligand-mediated arrestin-3 recruitment as well as affecting recruitment kinetics. Combined mutagenesis of all five of these residues was insufficient to fully abrogate interaction with arrestin-3, but further mutagenesis of negatively charged residues revealed additional structural components for the interaction with arrestin-3 within the C-terminal tail of the receptor. These elements consist of the acidic residues Glu³⁴¹, Asp³⁴⁸, and Asp³⁵⁵ located close to the phosphorylation sites. Receptor phosphorylation thus operates in concert with structural elements within the C-terminal tail of FFA4 to allow for the recruitment of arrestin-3. Importantly, these mechanisms of arrestin-3 recruitment operate independently from G_q/11 coupling, thereby offering the possibility that ligands showing stimulus bias could be developed that exploit these differential coupling mechanisms. Furthermore, this provides a strategy for the design of biased receptors to probe physiologically relevant signaling.