Growth cone interactions with a glial cell line from embryonic Xenopus retina

We have isolated a nonneuronal cell line from Xenopus retinal neuroepithelium (XR1 cell line). On the basis of immunocytochemical characterization using monoclonal antibodies generated in our laboratory as well as several other glial-specific antibodies, we have established that the XR1 cells are derived from embryonic astroglia. A monolayer of XR1 cells serves as an excellent substrate upon which embryonic retinal explants attach and elaborate neurites. This neurite outgrowth promoting activity appears not to be secreted into the medium, as medium conditioned by XR1 cells is ineffective in promoting outgrowth. Cell-free substrates were prepared to examine whether outgrowth promoting activity is also associated with the XR1 extracellular matrix (ECM). Substrates derived from XR1 cells grown on collagen are still capable of promoting outgrowth following osmotic shock and chemical extraction. This activity does not appear to be associated with laminin or fibronectin. Scanning electron microscopy was used to examine growth cones of retinal axons on XR1 cells and other substrates that supported neurite outgrowth. Growth cones and neurites growing on a monolayer of XR1 cells, or on collagen conditioned by XR1 cells, closely resemble the growth cones of retinal ganglion cells in vivo. A polyclonal antiserum (NOB1) generated against XR1 cells effectively and specifically inhibits neurite outgrowth on XR1-conditioned collagen. We therefore propose that neurite outgrowth promoting factors produced by these cells are associated with the extracellular matrix and may be glial specific.     

Microwave irradiation increases recovery of neuropeptides from brain tissues

The effect of focused high energy microwave treatment (MW) on brain concentrations and molecular forms of substance P, neurokinin A, neuropeptide Y, neurotensin, galanin and calcitonin gene-related peptide was investigated. Groups of rats were treated as follows: 1) MW, storage for 60 min at 22°C, 2) Decapitation, storage for 60 min at 22°C, 3) Decapitation, storage for 60 min at 22°C, MW treatment, 4) MW, decapitation, storage for 2 min at 22°C and 5) Decapitation, storage for 2 min at 22°C. Peptide concentrations were in all instances highest in the MW sacrificed groups. MW increased the concentration of intact peptides by rapid inhibition of peptidase activity and increase in peptide solubility/extractability.     

Detyrosination of tubulin is not correlated to cold-adaptation of microtubules in cultured cells from the Atlantic cod (Gadus morhua)

Summary Isolated cod brain microtubules from the cold-adapted Atlantic cod (Gadus morhua) have previously been shown to be highly detyrosinated, a post-translational modification of tubulin usually found in stable subsets of microtubules. In this study we found this was not restricted only to isolated brain microtubules. Microtubules in primary cultures of brain and skin cells were composed of both tyrosinated (Tyr)- and detyrosinated (Glu)-tubulin seen by immunocytochemistry. Immunoelectron microscopy of isolated microtubules showed that individual microtubules were composed of a mixture of Tyr- and Glu-tubulin. Leukocytes with extending lamellopodia contained only microtubules stained with the antibody against Tyr-tubulin, and isolated heart tubulin lacked both Tyr- and Glu-tubulin, suggesting that a relative high level of detyrosination is a characteristic of most, but not all, cod microtubules. Brain cell microtubules were more resistant to mitotic inhibitors than skin cell microtubules, but this was not correlated to a difference in detyrosination. Brain and skin cell microtubules were only partially disassembled when incubated at 0°C. Upon reassembly of microtubules at 12°C, microtubules were still made of mixtures of Tyr- and Glu-tubulin, indicating that detyrosination of assembled microtubules is rapid and/or that in cod cells, in contrast to mammalian cells, Glu-tubulin can reassemble to microtubules. Our data show that most cod microtubules are highly detyrosinated, but this is not the cause of their cold adaptation or drug stability.     

Inhibition of ATP-regulated K+-channels by a photoactivatable ATP-analogue in mouse pancreatic β-cells

The effects of a photoactivatable (DMNPE-caged) ATP-analogue on ATP-regulated K⁺-channels (K_ATP-channel) in mouse pancreatic β-cells were investigated using the inside-out patch configuration of the patch-clamp technique. The caged precursor caused a concentration-dependent reduction of channel activity with a K_i of 17 μM; similar to the 11 μM obtained for standard Mg-ATP. The small difference in the blocking capacity between the precursor and ATP is probably the reason why no change in channel activity was observed upon photolysis of the caged molecule and liberation of ATP. It is suggested that the part of the ATP molecule involved in the blocking reaction of the K_ATP-channel is not sufficiently protected in DMNPE-caged ATP making this compound unsuitable for studying the rapid kinetics of ATP-induced K_ATP-channel inhibition.     

Hydration-dependent far-infrared absorption in lysozyme detected using synchrotron radiation.

Using the National Synchrotron Light Source (NSLS) at Brookhaven far-infrared absorption in the frequency range 15-45 cm-1 was detected in samples of lysozyme at different hydrations and in water. The absorption is due to the presence of low-frequency (picosecond timescale) motion in the samples, such as are calculated in molecular dynamics simulations. The form of the transmission profile is temperature independent but varies significantly with the degree of hydration of the protein. At higher hydrations the profile resembles closely that of pure water in the region 20-45 cm-1. At a low hydration marked differences are seen with, in particular, the appearance of a transmission minimum at 19 cm-1. The possible origins of the hydration dependence are discussed. The results demonstrate the usefulness of long-wavelength synchrotron radiation for the characterisation of biologically-important low-frequency motions in protein samples.     

α-Glucosidase inhibition by flavonoids: an in vitro and in silico structure–activity relationship study

α-Glucosidase inhibitors are described as the most effective in reducing post-prandial hyperglycaemia (PPHG) from all available anti-diabetic drugs used in the management of type 2 diabetes mellitus. As flavonoids are promising modulators of this enzyme’s activity, a panel of 44 flavonoids, organised in five groups, was screened for their inhibitory activity of α-glucosidase, based on in vitro structure–activity relationship studies. Inhibitory kinetic analysis and molecular docking calculations were also applied for selected compounds. A flavonoid with two catechol groups in A- and B-rings, together with a 3-OH group at C-ring, was the most active, presenting an IC₅₀ much lower than the one found for the most widely prescribed α-glucosidase inhibitor, acarbose. The present work suggests that several of the studied flavonoids have the potential to be used as alternatives for the regulation of PPHG.