Structure of rapidly frozen gap junctions

The structure of gap junctions in the rabbit ciliary epithelium, corneal endothelium, and mouse stomach and liver was studied with the freeze-fracturing technique after rapid freezing to near 4 degrees K from the living state. In the ciliary epithelium, the connexons were randomly distributed, separated by smooth membrane matrix. In the corneal endothelium, both random and crystalline arrangements of the connexons were observed. In the stomach and liver, the connexons were packed but not crystalline. Experimental anoxia or lowered pH caused crystallization of the connexons within 20-30 min. In the ciliary epithelium, the effects of prolonged anoxia or low pH could not be reversed . In addition, invaginated or annular gap junctions increased in number, but their connexons were usually distributed at random. Rapid freezing thus demonstrates that gap junctions of different tissues are highly pleiomorphic in the living state, and this may explain their variations in structure after chemical fixation. The slow time-course and irreversibility of the morphological changes induced by prolonged anoxia or low pH suggest that connexon crystallization may be a long-term consequence rather than the morphological correlate of the switch to high resistance.     

Rapidly exchanging Ca2+ stores of non-muscle cells

The rapid and transient redistribution of calcium from intracellular stores is a key event of cell activation. The nature and molecular composition of intracellular Ca2+ stores of non-muscle cells are the object of intense investigation. In this paper, we review: (a) the experimental evidence in favor of the existence of intracellular, membrane-bound compartments specialized for uptake, storage and release of calcium, (b) the main protein components of rapidly exchanging Ca2+ stores, i.e. Ca2+ pump, intralumenal Ca2+ binding proteins (calsequestrin, calreticulin, etc.) and Ca2+ channels sensitive to either inositol 1,4,5-trisphosphate or Ca2+, caffeine and ryanodine, and (c) the relationship between Ca2+ stores and the endoplasmic reticulum.     

The effect of epidermal growth factor on membrane potential. Rapid hyperpolarization followed by persistent fluctuations

The effects of epidermal growth factor (EGF) on membrane potential were investigated in suspensions of the following three cell types endowed with a large complement of specific receptors: EGFR-T17 (a clone of mouse NIH-3T3 fibroblasts overexpressing EGF receptors); A431 and KB (two human carcinoma lines). In all these lines EGF induced a rapid and marked hyperpolarization constituted by an initial peak (in all three cell lines) and a subsequent sustained plateau phase, concomitant with the well-known increase of [Ca2+]i. The time course and phorbol ester inhibitability of the membrane potential effects were the same as for the [Ca2+]i response. Experiments with Na+-free and chloride-free media excluded a major role of the latter ions in the EGF-induced hyperpolarization. In contrast, experiments with high K+ media, with the monovalent cation ionophore gramicidin and with Ca2+-free media together with either a Ca2+ ionophore (ionomycin, in A431 and EGFR-T17), or an agonist (bradykinin, in A431) addressed to a receptor coupled to phosphoinositide hydrolysis, were consistent with the involvement of Ca2+-activated K+ channels. The EGF-induced hyperpolarization was completely blocked by the K+ channel blocker, quinidine, and unaffected by a variety of other drugs. Patch clamping of individual EGFR-T17 cells confirmed the initial hyperpolarization (from approximately -30 mV, the resting potential, to -60, -80 mV) was due to activation of an outward current. This initial hyperpolarization was followed by fluctuations (period approximately 1 min) persisting as long as the cells could be analyzed. Thus, the changes of membrane potential appear to be not only novel members of the group of early events triggered by EGF in target cells but also long-lasting effects of the growth factor, which continue for unexpectedly long periods of time after EGF application.     

Prevailingly cationic agmatine-based amphoteric polyamidoamine as a nontoxic, nonhemolytic, and "stealthlike" DNA complexing agent and transfection promoter

AGMA1, a prevailingly cationic amphoteric polyamidoamine obtained by polyaddition of (4-aminobutyl)guanidine (agmatine) to 2,2-bis(acrylamido)acetic acid, was studied as a potential DNA carrier and transfection promoter. Fluorescein-labeled AGMA1 was prepared by conjugation with fluorescein isothiocyanate and its cell uptake, blood permanence, and body distribution studied. In spite of its cationic character, AGMA1 is neither toxic nor hemolytic in the pH range 4.0-7.4, circulates for a long time in the blood without preferentially localizing in the liver, easily enters HT-29 cells, gives stable complexes with DNA, and is endowed with good transfection efficiency, suggesting the ability to transport in the cytoplasm a DNA payload without any measurable membranolytic activity. If compared with other transfection promoters, including polyamidoamines of different structures, AGMA1 is apparently endowed with a unique combination of desirable requirements for a nonviral DNA polymer carrier and warrants potential as a transfection agent in vivo.     

Breathing, locomotion and everything in-between. Proceedings of a symposium held at the 2nd International Congress of Respiratory Science. Bad Honnef, Germany. August 9-13, 2009

In the chick embryo at day 3, gas exchange occurs by diffusion and oxygen consumption (V?_O2) does not depend on the cardiovascular convection of O₂. Whether or not this is the case in hypoxia is not known and represents the aim of the study. The heart of chicken embryos at 72 h (stage HH18) was filmed through a window of the eggshell by a camera attached to a microscope. Stroke volume was estimated from the changes in heart silhouette between systole and diastole. V?_O2was measured by a closed system methodology. In normoxia, a decrease in temperature (T) from 39 to 31 °C had parallel depressant effects on V?_O2and HR. At 39 °C, a progressive decrease in O₂ lowered V?_O2; HR was maintained until the O₂ threshold of ～ 15%. In severe hypoxia (4% O₂) V?_O2and HR were, respectively, ～ 12% and ～ 62% of normoxia. At 32 °C the hypoxic threshold for HR was significantly lower. During constant hypoxia (7% O₂) V?_O2did not respond to T, while the HR response was preserved. Stroke volume changed little with changes in T or O₂, except at 6 and 4% O₂, when it decreased by ～ 20 and 30%. In embryos growth-retarded because of incubation in chronic hypoxia, V?_O2and HR responses to T and hypoxia were similar to those of normal embryos. We conclude that in the early embryo during hypoxia cardiovascular O₂ convection is not responsible for the drop in V?_O2. The generalised hypometabolic response, in combination with the extremely small cardiac V?_O2, probably explains the minor effects of hypoxia on cardiac activity.