Experimental depression of junctional membrane permeability in mammalian cell culture. A study with tracer molecules in the 300 to 800 dalton range

Summary Cell-to-cell junctional permeability in mammalian cell cultures was probed with a series of fluorescent tracers ranging 300 to 800 in molecular weight, during treatment with metabolic inhibitors, Ca-transporting ionophore, and carbon dioxide. Treatment with the combination of cyanide and iodoacetic acid (1–2mm each), but not with either one alone, caused reversible junctional blockade to all tracer molecular species, large and small. (Electrical coupling, however, persisted in a proportion of the junctions tested.) Treatment with the ionophore A23187 (2–10 m) or with CO₂ (an atmosphere of 100% CO₂ equilibrated with the medium) produced selective junctional blockade: transmission of a 688 and an 817-dalton tracer was generally blocked, while that of a 376-dalton tracer and, in certain conditions, that of a 559-dalton one, persisted. The junctional effect of the ionophore required the presence of Ca in the external medium; and effective junctional blockade by CO₂ required pretreatment in medium with high Ca concentration or, interchangeably, pretreatment in medium with high CO₂ concentration. In one cell type, prolonged exposure to medium with high Ca concentration alone sufficed to block transmission of the 688-dalton tracer. These effects are discussed in terms of the Ca hypothesis of junctional permeability regulation. In comparison with mammalian (or other vertebrate and invertebrate) organized tissues or with insect cell cultures, the mammalian cell cultures are more resistant to junctional blockade. This difference in transmission stability is discussed in terms of intracellular Ca-buffering capacities of the junctional locales; in particular, in terms of the electron-microscopic finding in the mammalian cultures of fine, bilateral cell processes connected by gap junctions.     

Intercellular communication and tissue growth: IX. Junctional membrane structure of hybrids between communication-competent and communication-incompetent cells

Summary The structure of the membrane junctions of the hybrid cell system, examined in the companion paper in respect to competence for communication through cell-to-cell membrane channels, is here examined by freeze-fracture electron microscopy. The junctions of the channel-competent parent cell and of the channel-competent hybrid cells present aggregates of intramembranous particles typical of gap junction; those of the channel-incompetent parent cell and channel-incompetent segregant hybrid cells do not. Competence for junctional communication and for gap junction formation are genetically related. The junctions of the intermediate hybrid cells with incomplete channel-competence (characterized by cell-to-cell transfer of small inorganic ions but not of fluorescein), present special intramembranous fibrillar structures instead of discrete gap-junctional particles. The possibility that these structures may constitute coupling elements with subnormal permeability is discussed in terms of incomplete dominance of the genetic determinants of gap junction.     

Hormonal regulation of cell junction permeability: Upregulation by catecholamine and prostaglandin E1

Summary By cellular activation with hormones, we test the proposition (Loewenstein, W.R.,Physiol. Rev. 61:829, 1981) that the permeability of cell junction is upregulated through elevation of the level of cyclic AMP. Cultured rat glioma C-6 cells, with -adrenergic receptors, and human lung WI-38 cells, with prostaglandin receptors, were exposed to catecholamine (isoproterenol) and prostaglandin E₁, respectively, while their junctions were probed with microinjected fluorescent-labelled mono-, di-, and triglutamate. Junctional permeability, as indexed by the proportion of cell interfaces transferring the probes, rose after the hormone treatments. The increase in permeability took several hours to develop and was associated with an increase in the number of gap-junctional membrane particles (freeze-fracture electron microscopy). Such interaction between hormonal and junctional intercellular communication may provide a mechanism for physiological regulation of junctional communication and (perhaps as part of that) for physiological coordination of responses of cells in organs and tissues to hormones.     

Spatial Generalization in Operant Learning: Lessons from Professional Basketball

In operant learning, behaviors are reinforced or inhibited in response to the consequences of similar actions taken in the past. However, because in natural environments the “same” situation never recurs, it is essential for the learner to decide what “similar” is so that he can generalize from experience in one state of the world to future actions in different states of the world. The computational principles underlying this generalization are poorly understood, in particular because natural environments are typically too complex to study quantitatively. In this paper we study the principles underlying generalization in operant learning of professional basketball players. In particular, we utilize detailed information about the spatial organization of shot locations to study how players adapt their attacking strategy in real time according to recent events in the game. To quantify this learning, we study how a make \ miss from one location in the court affects the probabilities of shooting from different locations. We show that generalization is not a spatially-local process, nor is governed by the difficulty of the shot. Rather, to a first approximation, players use a simplified binary representation of the court into 2 pt and 3 pt zones. This result indicates that rather than using low-level features, generalization is determined by high-level cognitive processes that incorporate the abstract rules of the game.     

The role of disjunction and postglacial population expansion on phylogeographical history and genetic diversity of the circumboreal plant Chamaedaphne calyculata

In the last decade a number of studies has illustrated quite different phylogeographical patterns amongst plants with a northern present‐day geographical distribution, spanning the entire circumboreal region and/or circumarctic region and southern mountains. These works, employing several marker systems, have brought to light the complex evolutionary histories of this group. Here I focus on one circumboreal plant species, Chamaedaphne calyculata (leatherleaf), to unravel its phylogeographical history and patterns of genetic diversity across its geographical range. A survey of 29 populations with combined analyses of chloroplast DNA (cpDNA), internal transcribed spacer (ITS) and AFLP markers revealed structuring into two groups: Eurasian/north‐western North American, and north‐eastern North American. The present geographical distribution of C. calyculata has resulted from colonization from two putative refugial areas: east Beringia and south‐eastern North America. The variation of chloroplast DNA (cpDNA) and ITS sequences strongly indicated that the evolutionary histories of the Eurasian/north‐western North American and the north‐eastern North American populations were independent of each other because of a geographical disjunction in the distribution area and ice‐sheet history between north‐eastern and north‐western North America. Mismatch analysis using ITS confirmed that the present‐day population structure is the result of rapid expansion, probably since the last glacial maximum. The AFLP data revealed low genetic diversity of C. calyculata (P = 19.5%, H = 0.085) over the whole geographical range, and there was no evidence of loss of genetic diversity within populations in the continuous range, either at the margins or in formerly glaciated and nonglaciated regions. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 761–775.     

Intramolecular relationships in cholinesterases revealed by oocyte expression of site-directed and natural variants of human BCHE.

Structure-function relationships of cholinesterases (CHEs) were studied by expressing site-directed and naturally occurring mutants of human butyrylcholinesterase (BCHE) in microinjected Xenopus oocytes. Site-directed mutagenesis of the conserved electronegative Glu441,Ile442,Glu443 domain to Gly441,Ile442,Gln443 drastically reduced the rate of butyrylthiocholine (BTCh) hydrolysis and caused pronounced resistance to dibucaine binding. These findings implicate the charged Glu441,Ile442,Glu443 domain as necessary for a functional CHE catalytic triad as well as for binding quinoline derivatives. Asp70 to Gly substitution characteristic of 'atypical' BCHE, failed to alter its Km towards BTCh or dibucaine binding but reduced hydrolytic activity to 25% of control. Normal hydrolytic activity was restored to Gly70 BCHE by additional His114 or Tyr561 mutations, both of which co-appear with Gly70 in natural BCHE variants, which implies a likely selection advantage for these double BCHE mutants over the single Gly70 BCHE variant. Gly70 BCHE variants also displayed lower binding as compared with Asp70 BCHE to cholinergic drugs, certain choline esters and solanidine. These effects were ameliorated in part by additional mutations or in binding solanidine complexed with sugar residues. These observations indicate that structural interactions exist between N' and C' terminal domains in CHEs which contribute to substrate and inhibitor binding and suggest a crucial involvement of both electrostatic and hydrophobic domains in the build-up of the CHE active center.     

Phosphorylation in vitro of Escherichia coli-produced 235R and 266R tumor antigens encoded by human adenovirus type 12 early transformation region 1A.

The tumor (T) antigens encoded by the human adenovirus early transforming region 1A (E1A) are gene regulatory proteins whose functions can immortalize cells. We have recently described the synthesis in Escherichia coli and the purification of the complete T antigens encoded by the adenovirus type 12 (Ad12) E1A 12S mRNA (235-residue [235R] T antigen) and 13S mRNA (266R T antigen). In this study, we show that the Ad12 E1A T antigens are extensively phosphorylated in Ad12-infected mammalian cells but are not phosphorylated in E. coli. Inasmuch as posttranslational phosphorylation at specific amino acid sites may be important for biological activity, we have studied the phosphorylation of the E. coli-produced T antigens in vitro by using a kinase activity isolated from cultured human KB cells. The kinase was purified about 300-fold and appears to be a cyclic AMP-independent, Ca2+-independent protein kinase requiring only ATP and Mg2+ for activity. To determine which amino acids are phosphorylated and whether phosphorylation in vitro occurs at the same amino acid sites that are phosphorylated in vivo, the Ad12 E1A T-antigen species synthesized by infected cells were metabolically labeled with 32Pi and compared with the E. coli-produced E1A T antigens labeled in vitro with [gamma-32P]ATP by using the partially purified kinase. Partial V8 proteolysis analysis gave similar patterns for in vivo- and in vitro-phosphorylated T antigen. Two-dimensional maps of tryptic phosphopeptides and of chymotryptic phosphopeptides suggested that mainly the same amino acid sites are phosphorylated in vitro and in vivo and that phosphorylation occurred at multiple sites distributed throughout the T-antigen molecule. Serine was the only amino acid that was phosphorylated both in vivo and in vitro, and, surprisingly, most serines appeared to be phosphorylated. The feasibility of faithfully phosphorylating T antigens in vitro suggests that the E. coli-produced Ad12 E1A 235R and 266R T antigens may prove useful for molecular studies on T-antigen function.     

How Recent History Affects Perception: The Normative Approach and Its Heuristic Approximation

There is accumulating evidence that prior knowledge about expectations plays an important role in perception. The Bayesian framework is the standard computational approach to explain how prior knowledge about the distribution of expected stimuli is incorporated with noisy observations in order to improve performance. However, it is unclear what information about the prior distribution is acquired by the perceptual system over short periods of time and how this information is utilized in the process of perceptual decision making. Here we address this question using a simple two-tone discrimination task. We find that the “contraction bias”, in which small magnitudes are overestimated and large magnitudes are underestimated, dominates the pattern of responses of human participants. This contraction bias is consistent with the Bayesian hypothesis in which the true prior information is available to the decision-maker. However, a trial-by-trial analysis of the pattern of responses reveals that the contribution of most recent trials to performance is overweighted compared with the predictions of a standard Bayesian model. Moreover, we study participants'' performance in a-typical distributions of stimuli and demonstrate substantial deviations from the ideal Bayesian detector, suggesting that the brain utilizes a heuristic approximation of the Bayesian inference. We propose a biologically plausible model, in which decision in the two-tone discrimination task is based on a comparison between the second tone and an exponentially-decaying average of the first tone and past tones. We show that this model accounts for both the contraction bias and the deviations from the ideal Bayesian detector hypothesis. These findings demonstrate the power of Bayesian-like heuristics in the brain, as well as their limitations in their failure to fully adapt to novel environments.