Coordination between enzyme specificity and intracellular compartmentation in the control of protein-bound oligosaccharide biosynthesis

This laboratory has developed schemes for the control of biosynthesis of N- and O-glycosyl oligosaccharides based on studies in cell-free systems of glycosyl-transferase substrate specificities. These schemes are based on assumptions that may not be universally correct. For example, we have ignored the possible compartmentation of reactions in different cells or in different organelles within a cell. Recent evidence has indicated that the Golgi apparatus has at least three functionally distinct regions (cis, medial and trans). The addition of galactosyl and sialyl residues to the antennae of complex and hybrid N-glycans probably occurs entirely within the trans-cisternae while the N-acetylglucosaminyl-transferases which initiate these antennae appear to be located in a denser region of the Golgi (cis and/or medial cisternae). We have constructed a modified scheme for the biosynthesis of the antennae of N-glycans. This scheme combines our substrate specificity data (H. Schachter, S. Narasimhan, P. Gleeson and G. Vella, 1983, Can. J. Biochem. Cell Biol., 61, 1049-1066) with compartmentation data. It provides a basis for understanding the control of glycoprotein synthesis in normal tissues and in certain lectin-resistant mutant cell lines.     

Phloem, transport between organs and long-distance signalling

Phloem plays a major role in carbohydrate partitioning in the plant. It also controls the redistribution of various metabolites such as amino acids, vitamins, hormones, and ions. The molecular mechanisms responsible for phloem loading and unloading have been particularly well characterised, with the identification of sucrose and polyol transporters. The discovery of the role of phloem in the long-distance translocation of macromolecules, proteins, mRNA and small RNA has modified our understanding of the regulation of the coordination of some developmental and adaptation processes. This review details recent results concerning the transport and long-distance signalling that take place in the phloem.     

Interactions between cadmium,selenium and glutathione peroxidase in rat testis

In rats given a minimal damaging dose of ¹⁰⁹CdCl₂ (0.011 mmole/kg, s.c.), a visible hemorrhagic response was evident after 48 h when testicular Cd uptake exceeded a level of approx. 150 ng/g. Glutathione peroxidase (GSH-Px) activity was elevated in homogenates of these damaged testes. In rats whose testes were not damaged, the Cd levels were below 150 ng/g and the GSH-Px activity was similar to that of control animals injected with sodium acetate. Rat testis cytosol was found to contain two different GSH-Px activities when assayed with cumene hydroperoxide. These could be separated by gel filtration chromatography. The larger species (GSH-Px A) was eluted in the void volume on Sephadex G-150 and incorporated ⁷⁵Se from Na₂⁷⁵SeO₃ given 4 weeks earlier. The smaller species, of approx. 42 000 molecular weight (MW) (GSH-Px B), did not incorporate ⁷⁵Se and could be distinguished from GSH-Px A by its insensitivity to cyanide (10 mM). CdCl₂ (1 mM) did not inhibit GSH-Px activity when added in vitro to GSH-Px A or B from testicular cytosol, or to purified GSH-Px isolated from ovine erythrocytes. When ¹⁰⁹CdCl₂ was given in vivo to rats injected 4 weeks previously with a tracer dose of Na₂⁷⁵SeO₃ or added in vitro to cytosol prepared from similarly labeled rats, Sephadex G-150 chromatography of cytosol showed that most of the ¹⁰⁹Cd was eluted in a major peak of 34 000 MW. Little or no ¹⁰⁹Cd was found in association with ⁷⁵Se (major peak 140 000 MW) or GSH-Px activity. When ¹⁰⁹CdCl₂ was injected into rats given an equimolar dose of Na₂⁷⁵SeO₃ 30 min previously, ¹⁰⁹Cd uptake in cytosol was increased and both ¹⁰⁹Cd and ⁷⁵Se was shifted into a peak of 110 000 MW.The ¹⁰⁹Cd-binding peak of approx. 30 000–34 000 MW was the major Cd-binding fraction in cytosol of 7-week-old rats but was not detectable in 4-week-old rats. Susceptibility of the testes to Cd did not correlate with the presence of this peak, however, since 4-week-old rats were occassionally damaged by CdCl₂.     

Interactions between hormone and redox signalling pathways in the control of growth and cross tolerance to stress

The ability of plants to respond to a wide range of environmental stresses is highly flexible and finely balanced through the interaction of hormonal plant growth regulators and the redox signalling hub, which integrates information from the environment and cellular metabolism/physiology. Plant hormones produce reactive oxygen species (ROS) as second messengers in signalling cascades that convey information concerning changes in hormone concentrations and/or sensitivity to mediate a whole range of adaptive responses. Cellular redox buffering capacity that is determined largely by the abundance of ascorbate has a profound influence on the threshold at which hormone signalling is triggered and on the interactions between different hormones. Other antioxidants such as glutathione, glutaredoxins and thioredoxins are also central redox regulators of hormone signalling pathways. The complex network of cross-communication between oxidants and antioxidants in the redox signalling hub and the different hormone signalling pathways maximises productivity under stress-free situations and regulates plant growth, development, reproduction, programmed cell death and survival upon exposure to stress. This interactive network confers enormous regulatory potential because it allows plants to adapt to changing and often challenging conditions, while preventing boom or bust scenarios with regard to resources, ensuring that energy is produced and utilised in a safe and efficient manner.     

Protein transport and compartmentation in yeast

Many newly synthesized proteins must be translocated across one or more membranes to reach their destination in the individual organelles or membrane systems. Translocation, mostly requiring an energy source, a signal on the protein itself, loose conformation of the protein and the presence of cytosolic and/or membrane receptor-like proteins, is often accompanied by covalent modifications of transported proteins. In this review I discuss these aspects of protein transport via the classical secretory pathway and/or special translocation mechanisms in the unicellular eukaryotic organism Saccharomyces cerevisiae.     

Auxin in action: signalling, transport and the control of plant growth and development

Hormones have been at the centre of plant physiology research for more than a century. Research into plant hormones (phytohormones) has at times been considered as a rather vague subject, but the systematic application of genetic and molecular techniques has led to key insights that have revitalized the field. In this review, we will focus on the plant hormone auxin and its action. We will highlight recent mutagenesis and molecular studies, which have delineated the pathways of auxin transport, perception and signal transduction, and which together define the roles of auxin in controlling growth and patterning.     

Calcium signalling: dynamics,homeostasis and remodelling

Ca2+ is a highly versatile intracellular signal that operates over a wide temporal range to regulate many different cellular processes. An extensive Ca2+-signalling toolkit is used to assemble signalling systems with very different spatial and temporal dynamics. Rapid highly localized Ca2+ spikes regulate fast responses, whereas slower responses are controlled by repetitive global Ca2+ transients or intracellular Ca2+ waves. Ca2+ has a direct role in controlling the expression patterns of its signalling systems that are constantly being remodelled in both health and disease.     

Intracellular compartmentation of glutathione in rabbit renal proximal tubules

The intracellular compartmentation of glutathione in rabbit renal proximal tubules was determined using digitonin, the non-ionic detergent Lubrol PX, and rapid centrifugation. Glutathione was distributed between two pools within the proximal tubules. The mitochondrial pool was the largest, containing 72 percent of the cellular glutathione while the cytoplasmic pool contained the remaining 28 percent. These results are in marked contrast to rat hepatocytes in which 85 percent of the cellular glutathione is cytoplasmic and only 15 percent is mitochondrial (1).     

Nitrate transport and compartmentation in cereal root cells

Measurement of cytosolic nitrate is one of the factors requiredfor the resolution of factors controlling nitrate uptake andassimilation in plants and for identifying likely nitrate transportmechanisms at both the plasma membrane and tonoplast. This paperreviews methods and reported measurements of cytosolic nitratein higher plants and concludes that nitrate-selective microelectrodesare the best approach. These microelectrodes have been usedto measure intracellular nitrate activities in barley and maizeroot cells. Triplebarrelled electrodes, incorporating a pH-sensingbarrel have been used to identify the compartmental locationof the nitrate-selective tip giving unequivocal estimates ofvacuolar and cytosolic nitrate activities. The microelectrodemeasurements are used to discuss the possible mechanisms ofnitrate transport at both the tonoplast and plasma membrane.The energetics of possible proton-coupled transport systemsare described and the feasibility of the mechanism is discussed. Key words: Cytosol, compartmentation, Hordeum vulgare L, nitrate, roots, Zea mays L     

Interactions between gut peptides and the central melanocortin system in the regulation of energy homeostasis

Genetic and pharmacological studies have shown that the central melanocortin system plays a critical role in the regulation of energy homeostasis. Animals and humans with defects in the central melanocortin system display a characteristic melanocortin obesity phenotype typified by increased adiposity, hyperphagia, metabolic defects and increased linear growth. In addition to interacting with long-term regulators of energy homeostasis such as leptin, more recent data suggest that the central melanocortin system also responds to gut-released peptides involved in mediating satiety. In this review, we discuss the interactions between these systems, with particular emphasis on cholecystokinin (CCK), ghrelin and PYY(3-36).     

Interactions between sterol biosynthesis genes in embryonic development of Arabidopsis

The sterol biosynthesis pathway of Arabidopsis produces a large set of structurally related phytosterols including sitosterol and campesterol, the latter being the precursor of the brassinosteroids (BRs). While BRs are implicated as phytohormones in post-embryonic growth, the functions of other types of steroid molecules are not clear. Characterization of the fackel (fk) mutants provided the first hint that sterols play a role in plant embryogenesis. FK encodes a sterol C-14 reductase that acts upstream of all known enzymatic steps corresponding to BR biosynthesis mutants. Here we report that genetic screens for fk-like seedling and embryonic phenotypes have identified two additional genes coding for sterol biosynthesis enzymes: CEPHALOPOD (CPH), a C-24 sterol methyl transferase, and HYDRA1 (HYD1), a sterol C-8,7 isomerase. We describe genetic interactions between cph, hyd1 and fk, and studies with 15-azasterol, an inhibitor of sterol C-14 reductase. Our experiments reveal that FK and HYD1 act sequentially, whereas CPH acts independently of these genes to produce essential sterols. Similar experiments indicate that the BR biosynthesis gene DWF1 acts independently of FK, whereas BR receptor gene BRI1 acts downstream of FK to promote post-embryonic growth. We found embryonic patterning defects in cph mutants and describe a GC-MS analysis of cph tissues which suggests that steroid molecules in addition to BRs play critical roles during plant embryogenesis. Taken together, our results imply that the sterol biosynthesis pathway is not a simple linear pathway but a complex network of enzymes that produce essential steroid molecules for plant growth and development.     

Glypicans shunt the Wingless signal between local signalling and further transport

The two glypicans Dally and Dally-like have been implicated in modulating the activity of Wingless, a member of the Wnt family of secreted glycoprotein. So far, the lack of null mutants has prevented a rigorous assessment of their roles. We have created a small deletion in the two loci. Our analysis of single and double mutant embryos suggests that both glypicans participate in normal Wingless function, although embryos lacking maternal and zygotic activity of both genes are still capable of transducing the signal from overexpressed Wingless. Genetic analysis of dally-like in wing imaginal discs leads us to a model whereby, at the surface of any given cell of the epithelium, Dally-like captures Wingless but instead of presenting it to signalling receptors expressed in this cell, it passes it on to neighbouring cells, either for paracrine signalling or for further transport. In the absence of dally-like, short-range signalling is increased at the expense of long-range signalling (reported by the expression of the target gene distalless) while the reverse is caused by Dally-like overexpression. Thus, Dally-like act as a gatekeeper, ensuring the sharing of Wingless among cells along the dorsoventral axis. Our analysis suggests that the other glypican, Dally, could act as a classical co-receptor.     

Interactions between muscle stem cells,mesenchymal-derived cells and immune cells in muscle homeostasis,regeneration and disease

Recent evidence has revealed the importance of reciprocal functional interactions between different types of mononuclear cells in coordinating the repair of injured muscles. In particular, signals released from the inflammatory infiltrate and from mesenchymal interstitial cells (also known as fibro-adipogenic progenitors (FAPs)) appear to instruct muscle stem cells (satellite cells) to break quiescence, proliferate and differentiate. Interestingly, conditions that compromise the functional integrity of this network can bias muscle repair toward pathological outcomes that are typically observed in chronic muscular disorders, that is, fibrotic and fatty muscle degeneration as well as myofiber atrophy. In this review, we will summarize the current knowledge on the regulation of this network in physiological and pathological conditions, and anticipate the potential contribution of its cellular components to relatively unexplored conditions, such as aging and physical exercise.     

Interactions between CRF, epinephrine, vasopressin and glucocorticoids in the control of ACTH secretion

The secretion of ACTH by corticotrophs in the anterior lobe of the rat pituitary gland is under the stimulatory influence of at least three receptors, namely that for peptidic CRF (corticotropin-releasing factor), vasopressin and alpha 1-adrenergic agents. CRF is a potent stimulator of cyclic AMP accumulation as well as adenylate cyclase activity in the rat adenohypophysis, thus suggesting an important role of cyclic AMP as mediator of CRF action on ACTH secretion. Vasopressin causes a 2-fold increase of the stimulatory effect of CRF on ACTH release in rat anterior pituitary cells in culture. The potentiating effects of vasopressin on CRF-induced ACTH release are accompanied by parallel changes of intracellular cyclic AMP levels. Vasopressin, while having no effect on basal cyclic AMP levels, causes a 2-fold increase in CRF-induced cyclic AMP accumulation without affecting the ED50 value of CRF action. ACTH secretion is also stimulated by a typical alpha 1-adrenergic receptor. Epinephrine causes a marked stimulation of ACTH release which is additive to that of CRF. Epinephrine, in analogy with vasopressin, although having no effect alone on basal cyclic AMP levels, causes a marked potentiation of CRF-induced cyclic AMP accumulation. Glucocorticoids cause a near-complete inhibition of epinephrine-induced ACTH secretion within 4 h with the following order of ED50 values: triamcinolone acetonide (0.2 nM) greater than dexamethasone (1.0 nM) much greater than cortisol (11 nM) greater than corticosterone (22 nM). Similar effects are observed for CRF- and vasopressin-induced ACTH release. Although the activity of the pituitary-adrenocortical axis in the rat is highly dependent upon sex steroids, 17 beta-estradiol, 5 alpha-dihydrotestosterone and the pure progestin R5020 have no detectable effect on basal or epinephrine-induced ACTH release, thus illustrating the high degree of specificity of glucocorticoids in their feedback control of ACTH secretion. Moreover, glucocorticoids have no effect on CRF-induced cyclic AMP accumulation, thus indicating that their inhibitory effect is exerted at a step following cyclic AMP accumulation.