Proliferation, neurogenesis and regeneration in the non-mammalian vertebrate brain

Post-embryonic neurogenesis is a fundamental feature of the vertebrate brain. However, the level of adult neurogenesis decreases significantly with phylogeny. In the first part of this review, a comparative analysis of adult neurogenesis and its putative roles in vertebrates are discussed. Adult neurogenesis in mammals is restricted to two telencephalic constitutively active zones. On the contrary, non-mammalian vertebrates display a considerable amount of adult neurogenesis in many brain regions. The phylogenetic differences in adult neurogenesis are poorly understood. However, a common feature of vertebrates (fish, amphibians and reptiles) that display a widespread adult neurogenesis is the substantial post-embryonic brain growth in contrast to birds and mammals. It is probable that the adult neurogenesis in fish, frogs and reptiles is related to the coordinated growth of sensory systems and corresponding sensory brain regions. Likewise, neurons are substantially added to the olfactory bulb in smell-oriented mammals in contrast to more visually oriented primates and songbirds, where much fewer neurons are added to the olfactory bulb. The second part of this review focuses on the differences in brain plasticity and regeneration in vertebrates. Interestingly, several recent studies show that neurogenesis is suppressed in the adult mammalian brain. In mammals, neurogenesis can be induced in the constitutively neurogenic brain regions as well as ectopically in response to injury, disease or experimental manipulations. Furthermore, multipotent progenitor cells can be isolated and differentiated in vitro from several otherwise silent regions of the mammalian brain. This indicates that the potential to recruit or generate neurons in non-neurogenic brain areas is not completely lost in mammals. The level of adult neurogenesis in vertebrates correlates with the capacity to regenerate injury, for example fish and amphibians exhibit the most widespread adult neurogenesis and also the greatest capacity to regenerate central nervous system injuries. Studying these phenomena in non-mammalian vertebrates may greatly increase our understanding of the mechanisms underlying regeneration and adult neurogenesis. Understanding mechanisms that regulate endogenous proliferation and neurogenic permissiveness in the adult brain is of great significance in therapeutical approaches for brain injury and disease.     

Evolution of the haem-haem interaction in vertebrate haemoglobins - a hypothesis

A hypothesis about the evolution of the haem-haem interaction in haemoglobins has been formulated on the basis of available functional and structural data. It emerges that this cooperative muchanism is not necessarily due solely to the higher levels of protomer aggregation, because it occurs only in haemoglobins having the distal histidine. It is thus proposed that this amino acid residue might have had a significant role for the development of low-oxygen-affinity haemoglobins in vertebrates.     

Myosin-linked calcium regulation in vertebrate smooth muscle.

By the use of a new procedure, actomyosin may be extracted in high yield and purity from fowl gizzard which exhibits a calcium-dependent actin-activated ATPase activity comparable to that of the parent myofibril-like preparation. Studies of this vertebrate smooth muscle actomyosin show that the regulation of the actin-myosin interaction is effected, as in molluscan muscles, by the myosin molecule itself and not by an actin-linked regulatory system, as found in vertebrate skeletal muscle.Thus, calcium-sensitive smooth muscle actomyosin is composed of only myosin, actin and tropomyosin, any troponin-like components being absent. Myosin is the only component that binds significant amounts of calcium and shows a calcium-dependent actin-activated ATPase activity in the presence of F-actin from either gizzard or rabbit skeletal muscle.The cross-reaction of gizzard thin filaments with skeletal muscle myosin produces an actomyosin whose actin-activated ATPase is calcium-insensitive, showing that smooth muscle thin filaments do not serve a regulatory function.The effect of Mg²⁺ and pH, and evidence for the involvement of one of the myosin light chains in calcium regulation are described and discussed.     

Haem-rotational disorder in monomeric allosteric cyano-Met insect haemoglobins monitored by resonance Raman spectroscopy

The haem-rotational disorder (insertion of haem into globin rotated about the alpha, gamma-meso axis by 180 degrees) has been investigated in the cyano-Met form of the monomeric allosteric insect haemoglobins, CTT III and CTT IV, by resonance Raman spectroscopy. The effect of haem disorder on the resonance Raman spectra has been observed in proto-IX, deutero-IX, and meso-IX CTTs. Most importantly, in the absence of overlapping vinyl vibrations, we have identified two Fe-C-N bending vibrations at 401 cm-1 and 422 cm-1 (pH 9.5) for 57Fe deutero-IX CTT IV ligated with 13C15N-, which are attributed to the two haem-rotational components. One Fe-C-N bending mode at 422 cm-1 shows a pH-induced shift to 424 cm-1 (pH 5.5) indicating the t----r conformational transition, whereas the other bending mode is pH-insensitive, representing a non-allosteric component. By replacing the unsymmetrical porphyrins with the "symmetrical" protoporphyrin-III we eliminate the haem disorder. Then, sharpening of the Fe-N epsilon(His) (at 313 cm-1) and Fe-CN (at 453 cm-1) stretching modes is observed and a single Fe-C-N bending mode (at 412 cm-1) appears. In cyano-Met proto-IX CTT III two vinyl bending vibrations at 412 cm-1 and 591 cm-1 assigned by deuteration of the vinyl groups also reflect the haem disorder. The 412 cm-1 vinyl vibration is intensity-enhanced via through-space coupling with one of the Fe-C-N bending modes (at 412 cm-1). In the cyano-Met form of proto-III CTT III this vinyl vibration is shifted to 430 cm-1 resulting in a dramatic drop in intensity. It is most likely that the specific vinyl-protein interaction at position 4 in one of the haem-rotational components is the origin of the coupling between the Fe-C-N and vinyl bending modes. The Fe-N epsilon(proximal His) and the Fe-CN stretching vibrations as well as the Fe-C-N bending vibration have been identified by 54Fe/57Fe and 13C15N/12C15N/13C14N/12C14N isotope exchange.     

Energetics of allosteric regulation in muscle pyruvate kinase.

The regulatory mechanism of rabbit muscle pyruvate kinase has been studied as a function of temperature in conjunction with phenylalanine, the allosteric inhibitor. The inhibitory effect of phenylalanine is modulated by temperature. At low temperatures, the presence of phenylalanine is almost inconsequential, but as the temperature increases so does the phenylalanine-dependent inhibition of the kinetic activity. In addition, the presence of phenylalanine induces cooperativity in the relation between velocity and substrate concentration. This effect is especially pronounced at elevated temperature. The kinetic data were analyzed using an equation that describes the steady-state kinetic velocity data as a function of five equilibrium constants and two rate constants. Van't Hoff analysis of the temperature dependence of the equilibrium constants determined by nonlinear curve fitting revealed that the interaction of pyruvate kinase with its substrate, phosphoenolpyruvate, is an enthalpy-driven process. This is consistent with an interaction that involves electrostatic forces, and indeed, phosphoenolpyruvate is a negatively charged substrate. In contrast, the interaction of pyruvate kinase with phenylalanine is strongly entropy driven. These results imply that the binding of phenylalanine involves hydrophobic interaction and are consistent with the basic concepts of strengthening of the hydrophobic effect with an increase in temperature. The effect of phenylalanine at high temperatures is the net consequence of weakening of substrate-enzyme interaction and significant strengthening of inhibitor binding to the inactive state of pyruvate kinase. The effects of salts were also studies. The results show that salts also exert a differential effect on the binding of substrate and inhibitor to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonsymbiotic haemoglobins in plants.

General aspects regarding the presence of nonsymbiotic haemoglobin in plants are presented with the emphasis on those related to its function. As it becomes apparent that the nonsymbiotic haemoglobins are widespread across the plant kingdom and that they represent a more primitive and evolutionary older form of the plant globin genes, the question of their function becomes more attractive. While the physiological functions of the symbiotic haemoglobins in plants are well understood, almost nothing is known about their nonsymbiotic predecessors. Therefore, the known and hypothetical functions of haemoglobins in various systems are described along with information concerning properties and the regulation of expression of the nonsymbiotic haemoglobins. Interestingly, a number of nonsymbiotic haemoglobins have been shown to be hypoxia-inducible. The spatial and temporal pattern of this induction in barley may suggest that it is an integral part of the plants response to limiting oxygen stress.     

Ribonucleotide reductases: the evolution of allosteric regulation.

Ribonucleotide reductases catalyze in all living organisms the production of the deoxyribonucleotides required for DNA replication and repair. Their appearance during evolution was a prerequisite for the transition from the "RNA world," where RNA sufficed for both catalysis and information transfer, to today's situation where life depends on the interplay among DNA, RNA, and protein. Three classes of ribonucleotide reductases exist today, widely differing in their primary and quaternary structures but all with a highly similar allosteric regulation of their substrate specificity. Here, I discuss the diversities between the three classes, describe their allosteric regulation, and discuss the evidence for their evolution. The appearance of oxygen on earth provided the likely driving force for enzyme diversification. From today's characteristics of the three classes, including their allosteric regulation, I propose that the anaerobic class III reductases with their iron-sulfur cluster and the requirement for S-adenosylmethionine for the generation of a glycyl protein free radical are the closest relatives to an ancestor ribonucleotide reductase.     

Cooperativity and allostery in aquaporin 0 regulation by Ca2+

Aquaporin 0 (AQP0) is essential for eye lens homeostasis as is regulation of its water permeability by Ca²⁺, which occurs through interactions with calmodulin (CaM), but the underlying molecular mechanisms are not well understood. Here, we use molecular dynamics (MD) simulations on the microsecond timescale under an osmotic gradient to explicitly model water permeation through the AQP0 channel. To identify any structural features that are specific to water permeation through AQP0, we also performed simulations of aquaporin 1 (AQP1) and a pure mixed lipid bilayer under the same conditions. The relative single-channel water osmotic permeability coefficients (p_f) calculated from all of our simulations are in reasonable agreement with experiment. Our simulations allowed us to characterize the dynamics of the key structural elements that modulate the diffusion of water single-files through the AQP0 and AQP1 pores. We find that CaM binding influences the collective dynamics of the whole AQP0 tetramer, promoting the closing of both the extracellular and intracellular gates by inducing cooperativity between neighboring subunits.     

The role of dynamics in allosteric regulation

The biomolecular conformational changes often associated with allostery are, by definition, dynamic processes. Recent publications have disclosed the role of pre-existing equilibria of conformational substates in this process. In addition, the role of dynamics as an entropic carrier of free energy of allostery has been investigated. Recent work thus shows that dynamics is pivotal to allostery, and that it constitutes much more than just the move from the 'T'-state to the 'R'-state. Emerging computational studies have described the actual pathways of allosteric change.     

Homotropic allosteric regulation in monomeric mammalian glucokinase

Glucokinase catalyzes the ATP-dependent phosphorylation of glucose, a chemical transformation that represents the rate-limiting step of glycolytic metabolism in the liver and pancreas. Glucokinase is a central regulator of glucose homeostasis as evidenced by its association with two disease states, maturity onset diabetes of the young (MODY) and persistent hyperinsulinemia of infancy (PHHI). Mammalian glucokinase is subject to homotropic allosteric regulation by glucose-the steady-state velocity of glucose-6-phosphate production is not hyperbolic, but instead displays a sigmoidal response to increasing glucose concentrations. The positive cooperativity displayed by glucokinase is intriguing since the enzyme functions as a monomer under physiological conditions and contains only a single binding site for glucose. Despite the existence of several models of kinetic cooperativity in monomeric enzymes, a consensus has yet to be reached regarding the mechanism of allosteric regulation in glucokinase. Experimental evidence collected over the last 45 years by a number of investigators supports a link between cooperativity and slow conformational reorganizations of the glucokinase scaffold. In this review, we summarize advances in our understanding of glucokinase allosteric regulation resulting from recent X-ray crystallographic, pre-equilibrium kinetic and high-resolution nuclear magnetic resonance investigations. We conclude with a brief discussion of unanswered questions regarding the mechanistic basis of kinetic cooperativity in mammalian glucokinase.     

Cooperativity in the dopamine beta-monooxygenase reaction. Evidence for ascorbate regulation of enzyme activity

The steady-state kinetic behavior of dopamine beta-monooxygenase (D beta M) has been examined over a 1000-fold range of ascorbate concentrations. Kinetic plots exhibit extreme curvature indicative of apparent negative cooperativity in the interaction of D beta M with ascorbate, with a calculated Hill coefficient of 0.15-0.30. The observed cooperativity is found to be independent of enzyme concentration and tyramine and oxygen concentrations, as well as the pH employed for the assay. Similar kinetic data have been obtained with both soluble and purified membrane-derived forms of enzyme. An investigation of the effect of the anion activator fumarate upon the observed kinetic patterns has demonstrated a conversion to a less cooperative kinetic pattern at low pH and high concentrations of fumarate. This phenomenon is attributed to an inhibitory binding of the structurally similar monoanionic species of fumarate to the ascorbate reductant site. A simple model has been used to assess the change in apparent Vmax and Km parameters with increased ascorbate concentrations. At all pH values examined, there is a dramatic decrease in the affinity of D beta M for ascorbate from a Km of approximately 0.05-0.10 mM (ascorbate concentration less than 1 mM) to Km greater than 10 mM at limiting ascorbate; at the same time there is a 3- to 4-fold increase in the limiting Vmax value. Several models have been considered to explain the observed activation of D beta M by high levels of ascorbic acid.     

The allosteric regulation of hexokinase C from amphibian liver.

A type C hexokinase (ATP:D-hexose-6-phosphotransferase EC 2.7.1.1) was partially purified from the liver of the frog Calyptocephalella caudiverbera. The enzyme is inhibited by glucose levels in the range of normal blood sugar concentrations. The extent of the inhibition by glucose depends on the concentration of ATP, being most marked between 1 and 5 mM ATP. Fructose, although a substrate, was not inhibitory of its own phosphorylation. The inhibitory effect of high glucose levels exhibited a strong, reversible pH dependence being most marked at pH 6.5. At pH 7.5 the inhibition by high glucose levels was a function of the enzyme concentration, the effect being stronger at high enzyme concentrations, whereas no inhibition was observed when assaying very diluted preparations. At all enzyme concentrations studied, high levels of glucose caused no inhibition at pH 8.5, whereas at pH 6.5 strong inhibition was always observed. Short times of photooxidation of hexokinase C as well as incubation with low concentrations of p-chloromercuribenzoate resulted in the loss of the inhibition by excess of glucose. Glucose-6-phosphate was found to be a strong inhibitor of hexokinase C but only at high glucose levels. The inhibitory effect of glucose-6-P follows sigmoidal kinetics at low (about 0.02 mM) glucose concentrations, the Hill coefficient being 2.3. The kinetics of the inhibition became hyperbolic at high (greater than 0.2 mM) glucose levels. These results suggest that the inhibition of hexokinase C by excess glucose is due to the interaction of glucose with a second, aldose-specific, regulatory site on the enzyme. The modification of the inhibitory effect by ATP, glucose-6-P, enzyme concentration, and pH, all of them at physiological levels, indicates a major role for hexokinase C in the regulation of glucose utilization by the liver.     

A model for the allosteric regulation of pH-sensitive enzymes.

1. In an enzyme that has two independent binding sites for a ligand, any inhibitor that binds solely to the free enzyme will give rise to positive co-operativity. 2. A model is considered for the allosteric control of enzymes by effectors in which their effects are mediated by ligand-induced perturbations of the ionization constants of a group or groups involved in the binding of substrate to the active site. 3. The model described offers a plausible explanation for the observation that the sigmoidal initial-rate curves reported for some regulatory enzymes are not expressed at all pH values where the enzyme is catalytically active.     

Changes in the expression and localization of cohesin subunits during meiosis in a non-mammalian vertebrate, the medaka fish

Until the onset of anaphase, sister chromatids are bound to each other by a multi-subunit protein complex called cohesin. Since chromosomes in meiosis behave differently from those in mitosis, the cohesion and separation of homologous chromosomes and sister chromatids in meiosis are thought to be regulated by meiosis-specific cohesin subunits. Actually, several meiosis-specific cohesin subunits, including Rec8, STAG3 and SMC1beta, are known to exist in mammals; however, there are no reports of meiosis-specific cohesin subunits in other vertebrates. To investigate the protein expression and localization of cohesin subunits during meiosis in non-mammalian species, we isolated cDNA clones encoding SMC1alpha, SMC1beta, SMC3 and Rad21 in the medaka and produced antibodies against recombinant proteins. Medaka SMC1beta was expressed solely in gonads, while SMC1alpha, SMC3 and Rad21 were also expressed in other organs and in cultured cells. SMC1beta forms a complex with SMC3 but not with Rad21, in contrast to SMC1alpha, which forms complexes with both SMC3 and Rad21. SMC1alpha and Rad21 were mainly expressed in mitotically dividing cells in the testis (somatic cells and spermatogonia), although their weak expression was detected in pre-leptotene spermatocytes. SMC1beta was expressed in spermatogonia and spermatocytes. SMC1beta was localized along the chromosomal arms as well as on the centromeres in meiotic prophase I, and its existence on the chromosomes persisted up to metaphase II, a situation different from that reported in the mouse, in which SMC1beta is lost from the chromosome arms in late pachytene despite its universal presence in vertebrates.