Possible roles for cell-to-substratum adhesion in neuronal morphogenesis

The role of cell-to-substratum adhesion in the initiation, elongation, and branching of axons from embryonic sensory neurons was investigated. Cells from sensory ganglia of 4–8-day-old chicken embryos were cultured on several substrata: including collagen; polyornithine-, polylysine-, and polyglutamate-coated surfaces, and tissue culture dishes. The air-blaster method was used to measure growth cone-substratum adhesion.Growth cones adhere much more strongly to polyornithine- or polylysine-coated surfaces and to the upper surfaces of glial cells than to tissue culture plastic. Axons, too, adhere tightly to these substrata, and are crooked, whereas on tissue culture plastic, axons are not adherent and are straight. The fraction of neurons that form axons and the rates of axonal elongation and branching are markedly increased when cells are cultured on polyornithine-coated dishes as compared to tissue culture dishes.This correlation of strong adhesion and enhanced neuronal morphogenesis suggests that adhesive interactions between the growth cone and the microenvironment in an embryo are crucial parts of the initiation and elongation of neuronal processes. Regulation of neuronal morphogenesis may be expressed through the physicochemical properties of the interacting cell surfaces and extracellular environment.     

Protein-derivatised glass coverslips for the study of cell-to-substratum adhesion

A simple, mild procedure is described for attaching proteins covalently to glass surfaces. The density of protein may be controlled and quantitated. The adhesive behavior of BHK cells on surfaces coated with con A is described and further applications of similar “model extracellular matrices” are suggested.     

Endosome-lysosome fusion at low temperature

Based on an initial study (Dunn, W. A., Hubbard, A. L., and Aronson, Jr., N. N. (1980) J. Biol. Chem. 255, 5971-5978), low temperature is often used to selectively inhibit fusion between endosomes and lysosomes. Here we have tried to characterize the nature of this inhibition. In addition to endocytic contents markers, we have used a covalent membrane marker to measure the interaction between endosomes and lysosomes over extended periods of time at low temperature. Mouse macrophage cells (P388D1) and human skin fibroblasts were enzymatically labeled with radioactive galactose to provide a covalent marker for plasma-membrane glycoconjugates. Subsequent endocytic membrane traffic for 24 h at 16 degrees C resulted in a significant transfer of membrane marker, as well as of endocytic contents marker, to high density lysosomes, as observed by subcellular fractionation. The kinetics of this transfer have been analyzed for macrophages using the membrane marker, horseradish peroxidase as fluid-phase, and iodinated acetyl low density lipoprotein as receptor-mediated endocytic contents marker. Transfer to lysosomes occurred only about 6 h after application of the respective marker at 16 degrees C. When transfer to lysosomes was initiated by 15 min preincubation at 37 degrees C, subsequent cooling to 16 degrees C did not inhibit ongoing transfer which continued with the same kinetics as when observed after the lag phase. These results show that low temperature delays an unidentified pre-fusion step, but does not inhibit endosome-lysosome fusion as such.     

Phased A-tracts bind to the alpha subunit of RNA polymerase with increased affinity at low temperature.

Previously we showed that the expression of a Clostridium perfringens phospholipase C gene (plc) is activated by promoter upstream phased A-tracts in a low temperature-dependent manner. In this paper we characterize the interaction between the alpha subunit of C. perfringens RNA polymerase and the phased A-tracts. Hydroxyl radical footprinting and fluorescence polarization assaying revealed that the alpha subunit binds to the minor grooves of the phased A-tracts through its C-terminal domain with increased affinity at low temperature. The result provides a molecular mechanism underlying the activation of the plc promoter by the phased A-tracts.     

Microfluorometric measurement at low temperature.

A cooling chamber for microfluorometric measurement is described, which allows to cool under a microscope a biological sample till 77 K and to measure it with an objective of a numerical aperture 0.6. From first experiments with BAO (5)-stained Tradescantia pollen it can be concluded, that experiments in this range of temperature open new aspects regarding the interpretation of microfluorometric phenomena.     

One-electron photooxidation of porphyrins at low temperature

The π-cation radicals of the metalloporphyrins magnesium octaethylporphyrin (MgOEP), magnesium tetraphenylporphyrin (MgTPP), and zinc tetraphenylporphyrin (ZnTPP), as well as the free base porphyrins of tetratolylporphyrin (H₂TTP) and tetraphenylporphyrin (H₂TPP) have been formed at liquid nitrogen temperatures in a rigid matrix of alkyl chloride glasses containing CCl₄ or 1,1,2,2-tetrachloroethane (TCE), following photolysis of the porphyrins with visible light. The reaction proceeds via electron transfer from the photoexcited porphyrin to the solvent molecules; the efficiency of thie electron transfer may be qualitatively evaluated in terms of electron tunneling in the solid matrices. This is the first report of the photochemical formation of a free base porphyrin π-cation radical species.     

Photoreaction of tyrosin-iodinated bacteriorhodopsin at low temperature

To elucidate the role of tyrosine residues in the shift of max and the light-driven proton pump of bacteriorhodopsin~ the photochemical reaction of tyrosine-iodinated bacteriorhodopsin (tyr-mod-bR) was investigated by low-temperature spectrophotometry. After 4–5 of 11 tyrosine residues of bacteriorhodopsin were iodinated, the meta-intermediate of tyr-mod-bR in 75% glycerol solution became so stable that its decay could be observed even at room temperature and i t was stable in the dark for several hours at –65°C.Four batho-intermediates were formed by irradiation with green light (500 nm) at –170°C. Like native bacteriorhodopsin, these batho-intermediates were photoreversible at –170°C. Four corresponding meta-intermediates were also formed by irradiation at –60°C. Using the difference spectra between meta-intermediates and tyr-mod-bR, the absorption spectra of four kinds of tyr-mod-bRs, batho-intermediates, and meta-intermediates were estimated. Each was at shorter wavelengths than that of its corresponding type in native bacteriorhodopsin. The results indicate that two or more tyrosine residues have some role in determining color in native bacteriorhodopsin.     

Is photosynthetic acclimation to low temperature controlled by capacities for storage and growth at low temperature? Results from comparative studies of grasses and trees

The ability of warm-grown leaves to acclimate their photosynthetic machinery to low, non-freezing temperature was compared for contrasting species of grasses and trees. All trees ( Betula pubescens , Salix sp. and Picea abies ), and young plants of one of the grasses ( Hordeum vulgare ) showed acclimation of photosynthesis while the other two grasses ( Phalaris arundinacea and Festuca ovina ) did not. It was those species that maintained leaf sugar concentrations essentially unchanged that showed acclimation. Trees maintained leaf sugar concentrations essentially unchanged by effectively converting leaf sugar surpluses into storage compounds. Grasses were, by comparison, less effective. However, very young plants of Hordeum maintained leaf sugar concentrations unchanged by continued growth rather than by increased storage. This diversity of low-temperature responses are discussed in relation to possible different priorities of trees and grasses: for grasses to undergo cold hardening by allowing sugars to rise, and for trees to store sugars to allow photosynthesis to operate independently of growth as growth varies with growth rhythm and air temperature.     

Genetic decrease in fatty acid unsaturation of phosphatidylglycerol increased photoinhibition of photosystem I at low temperature in tobacco leaves

Leaves of transgenic tobacco plants with decreased levels of fatty acid unsaturation in phosphatidylglycerol (PG) exhibited a slightly lower level of the steady state oxidation of the photosystem I (PSI) reaction center P700 (P700(+)) than wild-type plants. The PSI photochemistry of wild-type plants was only marginally affected by high light treatments. Surprisingly, all plants of transgenic lines exhibited much higher susceptibility to photoinhibition of PSI than wild-type plants. This was accompanied by a 2.5-fold faster re-reduction rate of P700(+) in the dark, indicating a higher capacity for cyclic electron flow around PSI in high light treated transgenic leaves. This was associated with a much higher intersystem electron pool size suggesting over-reduction of the PQ pool in tobacco transgenic lines with altered PG unsaturation compared to wild-type plants. The physiological role of PG unsaturation in PSI down-regulation and modulation of the capacity of PSI-dependent cyclic electron flows and distribution of excitation light energy in tobacco plants under photoinhibitory conditions at low temperatures is discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Synthesis of mammalian mitochondrial rRNA at low temperature

L cells incubated at 19 °C synthesize mitochondrial rRNA but not cytosol rRNA. In addition to 16 and 13S mitochondrial rRNA, mitochondrial RNA sedimenting at higher S values was also synthesized. The processing of mitochondrial rRNA is slower at 19 °C than at 37 °C.     

The preservation of ciliated protozoa at low temperature

Reasonable recovery after exposure to ?196 °C and after storage in liquid nitrogen refrigeration for as long as 4 years has been achieved with Tetrahymena pyriformis and with syngens 1 and 4 of Paramecium aurelia, although the percentage of cells surviving has usually been low. A procedure evolved with one stock may have to be altered for other stocks, which is not surprising, since the “species” T. pyriformis includes organisms probably more distantly separated evolutionarily than are fish and man. Attempts to explain the ability of relatively few cells to survive these conditions by resistance to salt, age of the cultures, etc. have so far been inconclusive and suggest interaction among the many variables.     

Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature

Solid-state NMR spectra of membrane proteins often show significant line broadening at cryogenic temperatures. Here we investigate the effects of several cryoprotectants to preserve the spectral resolution of lipid membranes and membrane peptides at temperatures down to ~200 K. Trehalose, glycerol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), and polyethylene glycol (PEG), were chosen. These compounds are commonly used in protein crystallography and cryobiology. ¹³C and ¹H magic-angle-spinning spectra of several types of lipid membranes show that DMSO provides the best resolution enhancement over unprotected membranes and also best retards ice formation at low temperature. DMF and PEG-400 show slightly weaker cryoprotection, while glycerol and trehalose neither prevent membrane line broadening nor prevent ice formation under the conditions of our study. Neutral saturated-chain phospholipids are the most amenable to cryoprotection, whereas negatively charged and unsaturated lipids attenuate cryoprotection. ¹³C–¹H dipolar couplings and ³¹P chemical shift anisotropies indicate that high spectral resolution at low temperature is correlated with stronger immobilization of the lipids at high temperature, indicating that line narrowing results from reduction of the conformational space sampled by the lipid molecules at high temperature. DMSO selectively narrowed the linewidths of the most disordered residues in the influenza M2 transmembrane peptide, while residues that exhibit narrow linewidths in the unprotected membrane are less impacted. A relatively rigid β-hairpin antimicrobial peptide, PG-1, showed a linewidth increase of ~0.5 ppm over a ~70 K temperature drop both with and without cryoprotection. Finally, a short-chain saturated lipid, DLPE, exhibits excellent linewidths, suggesting that it may be a good medium for membrane protein structure determination. The three best cryoprotectants found in this work—DMSO, PEG, and DMF—should be useful for low-temperature membrane-protein structural studies by SSNMR without compromising spectral resolution.     

Studies on the adaptation of influenza virus replicated at low temperature. III. Biochemical studies.

Five strains of influenza viruses A(H3N2) replicated at low temperature passaged in cotton rats were reisolated. The properties of these strains replicated at low temperature were compared before and after passage in susceptible animals to check the stability of some its markers. At the same time original viruses replicated at 37 degrees C--which are different in epidemiological potency--were compared. The following parameters being tested: NA activity, HA titers, heat inactivation NA and Ha, Michaelis constants and optimum pH. We observed some differences between strains both replicated at low temperature after passage in the susceptible animal organism and original viruses from 37 degrees C. Viruses replicated at low temperature from original epidemiostrain are really cold adapted and remained stable after passage in the animals when the others derived from no epidemic strain are not stable.