The inositol trisphosphate receptor regulates a 50-second behavioral rhythm in C. elegans.

The C. elegans defecation cycle is characterized by the contraction of three distinct sets of muscles every 50 s. Our data indicate that this cycle is regulated by periodic calcium release mediated by the inositol trisphosphate receptor (IP3 receptor). Mutations in the IP3 receptor slow down or eliminate the cycle, while overexpression speeds up the cycle. The IP3 receptor controls these periodic muscle contractions nonautonomously from the intestine. In the intestinal cells, calcium levels oscillate with the same period as the defecation cycle and peak calcium levels immediately precede the first muscle contraction. Mutations in the IP3 receptor slow or eliminate these calcium oscillations. Thus, the IP3 receptor is an essential component of the timekeeper for this cycle and represents a novel mechanism for the control of behavioral rhythms.     

The molecular structure of the receptor-ionophore complex at the neuromuscular junction.

A general theory of the molecular structure of receptors for transmitters based only on protein has been presented elsewhere (Smythies, 1974a,b). The acetylcholine receptor at the neuromuscular junction is postulated in particular to be based on a Kusnetsov-Ghokov grid with four sequencestwo “primary” chains A-x-B-cys-A-x-B where A = arg or lys and B = glu or phosphoser and two “secondary” chains of sequence -gly-x-gly-pro-x-ile-cys-asp-x- forming a symmetrical receptor cup of rectangular form. The present paper extends the model to include the gate over the adjacent ionophore (or “ion conductance modulator”: ICM) and the linking mechanism from receptor to gate. These are postulated to consist of a second Kusnetsov-Ghokov grid generated by a third “primary” chain along the side that covers the orifice to the ion conducting channel. The action of ACh is postulated to be to displace an hydrated Ca⁺⁺ ion from the receptor cup and to disrupt the AB rungs in the receptor grid. The middle primary chain then slides 14 Å and the AB links reform. This replaces a bulky amino acid pair normally blocking the ion channel by a less bulky amino acid pair and so hydrated ions can be transmitted. It is further postulated that snake neurotoxins (ACh blockers) in a specified conformation bind mainly to the ionophore grid and prevent the sliding filament mechanism from opening; whereas the snake “cardiotoxins” (ACh agonists)—in a specified conformation—bind to the same sliding filament mechanism in its “open” ionophore gate and prevent it being closed: and histrionicotoxin binds to the same open “gate” but blocks it physically. The hypothesis may rigorously be tested by experiment as it makes detailed predictions on the X-ray structure of the snake neurotoxins and cardiotoxins.     

What is a cladogram and what is not?

The origins and meanings of “cladogram” are reviewed. Traditionally, “cladogram” has been defined as a graphical representation of an empirical hypothesis of relationships among taxa, based on evidence from synapomorphies alone. Disturbingly, numerous recent authors treat “cladogram” as synonymous with “dendrogram” and do not appreciate the particular methodological connotations of the former term. This is lamented.     

The case for reverse translation.

The theoretical and experimental evidence for a “reverse translation” mechanism in animal cells is reviewed. Mekler's (1967) theory is presented as the most likely means for reverse translation. This theory is shown to be consistent with the postulate of the “central dogma” that molecular information does not pass out of protein molecules once it has gotten in.The importance of nucleic acid changes in: (1) the immune response, (2) evolution, (3) cancer, (4) cell differentiation, and (5) learning in animal brains is mentioned and each topic is related to the reverse translation hypothesis. Because of the intense research effort in immunology, the experimental data which indicate an active role for antigen in antibody formation and the data indicating the importance of RNA in the immune response are dealt with more thoroughly.     

Mort ou persistance du darwinisme ? Regard d’un épistémologue

The article examines why evolutionary biologists have been haunted by the question whether they are “Darwinian” or “non-Darwinian” ever since Darwin's Origin of species. Modern criticisms addressed to Darwinism are classified into two categories: those concerning Darwin's hypothesis of “descent with modification” and those addressed to the hypothesis of natural selection. In both cases, although the particular models that Darwin proposed for these two hypotheses have been significantly revised and expanded, Darwin's general framework has constrained and canalized evolutionary research, in the sense that it has settled an array of possible theoretical choices. Gould's changing attitudes regarding Darwinism is taken as a striking illustration of this interpretation.     

Driving Protein Conformational Cycles in Physiology and Disease: “Frustrated” Amino Acid Interaction Networks Define Dynamic Energy Landscapes

A general framework by which dynamic interactions within a protein will promote the necessary series of structural changes, or “conformational cycle,” required for function is proposed. It is suggested that the free-energy landscape of a protein is biased toward this conformational cycle. Fluctuations into higher energy, although thermally accessible, conformations drive the conformational cycle forward. The amino acid interaction network is defined as those intraprotein interactions that contribute most to the free-energy landscape. Some network connections are consistent in every structural state, while others periodically change their interaction strength according to the conformational cycle. It is reviewed here that structural transitions change these periodic network connections, which then predisposes the protein toward the next set of network changes, and hence the next structural change. These concepts are illustrated by recent work on tryptophan synthase. Disruption of these dynamic connections may lead to aberrant protein function and disease states.     

Autophosphorylation of inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP3) releases internal stores of calcium by binding to a specific membrane receptor which includes both the IP3 recognition site as well as the associated calcium channel. The IP3 receptor is regulated by ATP, calcium, and phosphorylation by protein kinase A, protein kinase C, and calcium/calmodulin-dependent protein kinase II. Its cDNA sequence predicts at least two consensus sequences where nucleotides might bind, and direct binding of ATP to the IP3 receptor has been demonstrated. In the present study, we demonstrate autophosphorylation of the purified and reconstituted IP3 receptor on serine and find serine protein kinase activity of the IP3 receptor toward a specific peptide substrate. Several independent purification procedures do not separate the IP3 receptor protein from the phosphorylating activity, and many different protein kinase activators and inhibitors do not identify protein kinases as contaminants. Also, renaturation experiments reveal autophosphorylation of the monomeric receptor on polyvinylidene difluoride membranes.     

Peptide-membrane interactions and a new principle in quantitative structure-activity relationships.

Ideas about “active conformations” of peptides as the basis of their biological action are reviewed and put into perspective with recent work on the possible role of the lipid phase of the target cell membrane in mediating receptor subtype selectivity.     

Bliss points vs. minimum needs: Tests of competing motivational models

This paper tests two competing hypotheses concerning the motivational forces underlying concurrent choice behavior: a generalized version of Staddon's minimum-distance hypothesis, which characterizes behavior in terms of minimizing the distance to a “bliss” point, and a generalized minimum-needs hypothesis, which emphasizes meeting minimum survival requirements first, after which the organism is free to allocate behavior in any fashion desired. The models specify distinctly different preference structures. The generalized minimum-needs hypothesis is shown to provide a superior fit to molar choice data from experiments involving food and fluid consumption.     

Second messenger imbalance hypothesis of schizophrenia.

Based on the results of studies on intracellular signaling in platelets of schizophrenics, an imbalance of the second messenger system is proposed: Diacylglycerol (DG), which activates protein kinase C (PKC), was increased, while adenylate cyclase (AC)-cAMP function was decreased. It is proposed that the increased DG/PKC function may entail a decrease in inositol 1,4,5-trisphosphate (IP3)/Ca2+ function and lowering of phosphoinositide turnover. If such a pathological intracellular signaling takes place in the brain, it may cause a distorted balance of protein activation via phosphorylation in neurons, resulting in some of the deficits of schizophrenia. Neuroleptics have been reported to antagonize the above-mentioned pathological processes of intracellular signaling. The imbalance hypothesis of the DG/PKC pathway and AC-cAMP pathway is not inconsistent with the dopamine hypothesis of schizophrenia, because dopamine receptor stimulation is indirectly related to reduction in IP3/Ca2+ function and lowering of phosphoinositide metabolism.     

DIATOM MINERALIZATION OF SILICIC ACID. II. REGULATION OF Si (OH)4 TRANSPORT RATES DURING THE CELL CYCLE OF NAVICULA PELLICULOSA1

Synchronized populations of Navicula pelliculosa (Bréb.) Hilse show a 10-fold increase in Si(OH)₄ transport rate during traverse through the cell division cycle. The transport activity pattern is similar to a “peak enzyme.” Kinetic analysis showed there was a significant change in K_s values, indicating increased “affinity” for Si(OH)₄ as cells neared maximal uptake rates. However, the dramatic changes in transport rate at various cell cycle stages were also reflected by alterations in the V_max, values of the transport process, suggesting a change in the number of functional transport “sites” in the plasma membrane. Cells in the wall forming stage, arrested from further development by Si(OH)₄ deprivation, maintained high transport rates for as long as 7 h. The rates decreased rapidly if protein synthesis were blocked or if Si(OH)₄ was added, the latter allowing the cells to traverse the rest of the cycle. The half-life of the transport activity ranged from 1.0 to 2.2 h when protein synthesis was inhibited at various cell cycle stages and during the natural decline of activity late in the cycle. The transport system appears to be metabolically unstable as is typical for a “peak protein.” The rise in transport rate through the cell cycle did not depend on the presence of Si(OH)₄ in the medium; therefore, the transport system does not appear to be induced by its substrate. The rise in transport is also observed in L:D synchronized cells developing in the presence of Si(OH)₄; neither does the transport system appear to be derepressed. The transport rate was strongly cell cycle-stage dependent; the data appeared to fit the “dependent pathway” model proposed by Hart-well to explain oscillations in enzyme synthesis during the cell cycle.     

Inositol hexakisphosphate receptor identified as the clathrin assembly protein AP-2.

To clarify the function of the receptor binding protein for inositol hexakisphosphate (IP6), we obtained a partial amino acid sequence from the purified protein and a partial nucleotide sequence from a cDNA clone of the gene. The sequences are essentially identical to those of the alpha-subunit of the clathrin assembly protein AP-2. The IP6 receptor protein analyzed by SDS-PAGE contains a series of subunits which are the same as those of AP-2. Antibodies to AP-2 react with the IP6 receptor protein in immunoblot analysis.     

A hypothesis on the role of immune RNA in antibody variability

The present hypothesis on the mechanism of antibody variability considers immune RNA (IMRNA) as a relatively independent “entity”. After having been transcribed in ontogenesis, as a result of stimulation of primordial multipotent “master cells” by primordial antigens, IMRNA behaves like an RNA virus genome. It controls only the variable part of immunoglobulin chains, proliferates and is transferred from committed to uncommitted cells, directly or using the macrophage as an “intermediate host”. During IMRNA proliferation “mutant” IMRNAs appear and control the synthesis of antibody variable regions of new specificity. The IMRNAs are reverse transcribed and inserted into DNA, chromosomal or extrachromosomal, near the gene controlling the constant part, and a complete CV gene is formed. The selective pressures which assure a relative constancy of IMRNA, so that only changes in the so-called hypervariable region are “tolerated” might be: the recognition by replicase, the insertion device, and the antigen-surface immunoglobulin-membrane receptor interaction. Arguments from immunology and from other fields of biology are brought in support at this hypothesis, and experimental approaches are suggested.     

Protein 4.1N does not interact with the inositol 1,4,5-trisphosphate receptor in an epithelial cell line

Cytosolic Ca2+ regulates a variety of cell functions, and the spatial patterns of Ca2+ signals are responsible in part for the versatility of this second messenger. The subcellular distribution of the inositol 1,4,5-trisphosphate receptor (IP3R) is thought to regulate Ca2+-signaling patterns but little is known about how the distribution of the IP3R itself is regulated. Here we examined the relationship between the IP3R and the cytoskeletal linker protein 4.1N in the polarized WIF-B cell line because protein 4.1N regulates targeting of the type I IP3R in neurons, but WIF-B cells do not express this cytoskeletal protein. WIF-B cells expressed all three isoforms of the IP3R, and each isoform was distributed throughout the cell. These cells did not express the ryanodine receptor. Photorelease of microinjected, caged IP3 induced a rapid rise in cytosolic Ca2+, but the increase began uniformly throughout the cell rather than at a specific initiation site. Expression of protein 4.1N was not associated with redistribution of the IP3R or changes in Ca2+-signaling patterns. These findings are consistent with the hypothesis that the subcellular distribution of IP3R isoforms regulates the formation of Ca2+ waves, and the finding that interactions between protein 4.1N and the IP3R vary among cell types may provide an additional, tissue-specific mechanism to shape the pattern of Ca2+ waves.     

The mobile receptor hypothesis and “cooperativity” of hormone binding. Application to insulin

The mobile receptor hypothesis has been proposed to describe the process by which hormone receptor binding initiates a biological response; it states that receptors, which can diffuse independently in the plane of the membrane, reversibly associate with effectors to regulate their activity. The affinity for effector is greater when the receptor is occupied by hormone.A mathematical expression of the mobile receptor hypothesis is used to show that: (1) The predicted kinetics of hormone receptor binding may be indistinguishable from “negative cooperativity”. (2) Receptor occupancy and biological response may be coupled in a non-linear fashion.By choosing specific parameters, most of the existing data on insulin binding and biological responses can be explained in terms of the mobile receptor hypothesis. Thus, the following are easily explained: (1) A single homogeneous receptor may appear kinetically to be composed of two classes (of high and low affinity) of receptors. (2) Occupancy of the apparent class of high affinity receptors is related linearly to the biological response. (3) The same receptor in different tissues may appear to have different affinity. (4) The binding of different biologically active insulin analogues may exhibit different degrees of “cooperatively.” These considerations may also be pertinent to intepretations of other hormone-receptor systems and of various ligand-macromolecule interactions.     

The effects of peripherally injected serotonin on feeding and locomotor activities in carp Cyprinus carpio L.

The influence of intraperitoneal (IP) and intramuscular (IM) injections of serotonin (5-hydroxytryptamine or 5-HT, 10 μg/g body weight) on a number of parameters of feeding behavior and locomotor activity in carp Cyprinus carpio L. has been investigated. It was shown that exogenous serotonin decreased various parameters of feeding and locomotor activities, and IM injections caused stronger inhibitory effect than IP injections. IP administration of this biogenic amine reduced the food intake in fishes of different age groups, induced an increase of the search reaction time (the latency to leave the starting chamber after its front wall was raised, or latency period for feeding of fish) in carp fingerlings in the experiments with “single” feeding. IM injections significantly lowered food intake of carp fingerlings in 1, 5 and 53 h, two other parameters—during all period of observation. In the experiments with “group” feeding food intake, duration of “group” feeding and total duration of feeding decreased during all period of observation after IM administration and in 1 h after IP injections only. Duration of “single” feeding and locomotor activity were changed less distinctly. The strongest effect of serotonin (up to 100%) was shown for duration of “group” feeding. It was supposed that inhibitory effects of exogenous serotonin on feeding and locomotor activities in carps were caused by its peripheral effects as well as by partial involving of central effect.     

Modelling of simple and complex calcium oscillations. From single-cell responses to intercellular signalling.

This review provides a comparative overview of recent developments in the modelling of cellular calcium oscillations. A large variety of mathematical models have been developed for this wide-spread phenomenon in intra- and intercellular signalling. From these, a general model is extracted that involves six types of concentration variables: inositol 1,4,5-trisphosphate (IP3), cytoplasmic, endoplasmic reticulum and mitochondrial calcium, the occupied binding sites of calcium buffers, and the fraction of active IP3 receptor calcium release channels. Using this framework, the models of calcium oscillations can be classified into 'minimal' models containing two variables and 'extended' models of three and more variables. Three types of minimal models are identified that are all based on calcium-induced calcium release (CICR), but differ with respect to the mechanisms limiting CICR. Extended models include IP3--calcium cross-coupling, calcium sequestration by mitochondria, the detailed gating kinetics of the IP3 receptor, and the dynamics of G-protein activation. In addition to generating regular oscillations, such models can describe bursting and chaotic calcium dynamics. The earlier hypothesis that information in calcium oscillations is encoded mainly by their frequency is nowadays modified in that some effect is attributed to amplitude encoding or temporal encoding. This point is discussed with reference to the analysis of the local and global bifurcations by which calcium oscillations can arise. Moreover, the question of how calcium binding proteins can sense and transform oscillatory signals is addressed. Recently, potential mechanisms leading to the coordination of oscillations in coupled cells have been investigated by mathematical modelling. For this, the general modelling framework is extended to include cytoplasmic and gap-junctional diffusion of IP3 and calcium, and specific models are compared. Various suggestions concerning the physiological significance of oscillatory behaviour in intra- and intercellular signalling are discussed. The article is concluded with a discussion of obstacles and prospects.     

Mitochondrial groups of germinal cells of teleost Cyprinidae. II. High resolution autoradiographic study of the incorporation of 3H phenylalanine and 3H uridine

A high resolution autoradiographic study of the incorporation of tritiated uridine and amino acids by the mitochondrial groups, which are typical of most of germ cells at the beginning of gametogenesis, has been made on tench spermatocytes. By this technique, we confirm the partially proteinacious composition of the intermitochondrial “cement”; and for the first time, the presence of RNA in the “cement” is demonstrated by autoradiography. Moreover, a study of the kinetics of the incorporation of both precursors makes likely the hypothesis that at least a part of the “cement” derived from nucleocytoplasmic transfer. Since the biogenesis of mitochondria results from the complementary functioning of the two protein synthesic systems, the cytoplasmic one, which is preponderant, and the mitochondrial one, the mitochondrial groups seem to be the direct visualization of the contribution of the nuclear genome to the edification of new mitochondria.     

RADIOSENSITIVITY,RNA, AND PROTEIN METABOLISM OF “LEAKY” AND ARRESTED CELLS IN SUNFLOWER ROOT MERISTEMS (HELIANTHUS ANNUUS)

Two cell populations in sunflower root meristems are described. Most cells stop in G1 after being cultured in sucrose-deficient medium, but “leaky” cells continue through DNA synthesis and stop in G2. A comparison of “leaky” and arrested cells is reported on the basis of radiosensitivity, and cytological and biochemical responses to metabolic inhibitors. “Leaky” cells are randomly distributed throughout primary meristematic tissues. They are not inhibited from initiating DNA synthesis by exposure to doses of γ-irradiation ranging from 300–7200 R; arrested cells, depending upon the dose, are inhibited partially or completely. “Leaky” cells do, however, show a dose-dependent mitotic delay in G2, which is the same as arrested cells. Treatment with puromycin and actidione does not inhibit “leaky” cells from initiating DNA synthesis but does inhibit them from mitosis. Arrested cells are inhibited from advancing to S and M by both inhibitors. Also, puromycin and actidione cause a decrease in protein and RNA synthesis, demonstrating a possible protein dependent RNA synthesis necessary for cell cycle progression. Actinomycin D (10 μg/ml) inhibits neither “leaky” nor arrested cells from entering S and M. At 30 μg/ml, however, arrested cells are partially inhibited. “Leaky” cell metabolism is unique in preparation for and initiation of DNA synthesis but similar to that of the remaining cells of the meristem in terms of requirements for progression through the rest of the mitotic cycle.