Structure-function relationships in glucagon. Re-evaluation of glucagon-(1-21)

Glucagon-(1-21) was prepared fully synthetically as well as by carboxypeptidase A digestion of natural porcine glucagon. Neither of the two preparations had glucagon agonistic effects with regard to receptor binding or adenylate cyclase activation in purified rat liver plasma membranes. Nor did these preparations contain lipolytic activity in isolated free fat cells. A preliminary batch of glucagon-(1-21) prepared by carboxypeptidase A digestion did, however, contain 1-2% glucagon bioactivity. This activity was separated from glucagon-(1-21) by high-performance liquid chromatography and quantitatively recovered in four minor hind peaks which eluted close to but not in a position identical to the elution position of native glucagon.     

Glucagon1-6 binds to the glucagon receptor and activates hepatic adenylate cyclase.

A fragment of glucagon encompassing its first six NH2-terminal residues (His-Ser-Gln-Gly-Thr-Phe) binds to the glucagon receptor and stimulates adenylate cyclase activity in rat liver plasma membranes. Glucagon1-6 is a partial agonist since it stimulates, at saturating concentrations, to the extent of 75% of the maximal activity given by the native hormone. The binding affinity and potency of glucagon1-6 are 0.001% the native hormone. Discussed are the implications of these findings on the structure-function relationships required for the action of glucagon and for preparing clinically useful analogs of the hormone.     

A quantitative investigation of structure-function relationships in a tendon fascicle model

These studies sought to investigate quantitative relationships between the complex composite structure and mechanical properties of tendon. The isolated mouse tail tendon fascicle was chosen as an appropriate model for these so-called "structure-function" investigations. Specifically, collagen fibril diameters and mechanical properties were measured in fascicles from immature (3 week) control, adult (8 week) control, and adult (8 week) MovI3 transgenic mice. Results demonstrated a moderate correlation between mean fibril diameter and fascicle stiffness (r = 0.73, p = 0.001) and maximum load (r = 0.75, p < 0.001), whereas a weak correlation with fascicle modulus (r = 0.39, p = 0.11) and maximum stress (r = 0.48, p = 0.04). An analysis of pooled within-group correlations revealed no strong structure-function trends evidenced at the local or group level, indicating that correlations observed in the general structure-function analyses were due primarily to having three different experimental groups, rather than significant correlations of parameters within the groups.     

Occluding junction structure-function relationships in a cultured epithelial monolayer

Electrical circuit analysis was used to study the structural development of occluding junctions (OJs) in cultured monolayers composed to T84 cells. The magnitude of the increments in transepithelial resistance predicted by such analysis was compared with the magnitude of the measured increments in resistance. Confluent sheets of epithelial cells were formed after cells were plated at high density on collagen-coated filters. Using Claude's OJ strand count-resistance hypothesis (1978, J. Membr. Biol. 39:219-232), electrical circuit analysis of histograms describing OJ strand count distribution at different time points after plating predicted that junctional resistance should rise in a proportion of 1:21:50 from 18 h to 2 d to 5 d. This reasonably paralleled the degree of rise in transepithelial resistance over this period, which was 1:29:59. The ability to predict the observed resistance rise was eliminated if only mean strand counts were analyzed or if electrical circuit analysis of OJ strand counts were performed using an OJ strand count-resistance relationship substantially different from that proposed by Claude. Measurements of unidirectional fluxes of inulin, mannitol, and sodium indicated that restriction of transjunctional permeability accounted for the observed resistance rise, and that T84 junctional strands have finite permeability to molecules with radii less than or equal to 3.6 A but are essentially impermeable to molecules with radii greater than or equal to 15 A. The results suggest that general correlates between OJ structure and OJ ability to resist passive ion flow do exist in T84 monolayers. The study also suggests that such correlates can be obtained only if OJ structural data are analyzed as an electrical circuit composed of parallel resistors.     

Obfuscation of allosteric structure-function relationships by enthalpy-entropy compensation.

The pH and temperature dependence of the allosteric properties of phosphofructokinase (PFK) from Bacillus stearothermophilus have been studied from 5 to 9 and 6 to 40 degrees C, respectively. Throughout this pH and temperature range the allosteric ligands MgADP and phospho(enol)pyruvate (PEP) have no effect on kcat. The dissociation constants of the substrate, fructose 6-phosphate, and the allosteric ligands, as well as the absolute value of the coupling free energies between these ligands, all increase when the pH is raised, indicating that the inhibition by PEP and the activation by MgADP increase despite each ligand's somewhat lower affinity. However, the constituent coupling enthalpies and entropies substantially diminish in absolute value as pH is increased, suggesting that the magnitudes of molecular perturbations engendered by the binding of allosteric ligands do not correlate with the magnitudes of the functional consequences of those perturbations. Temperature and pH exert their influence on the observed allosteric behavior by changing the relative contributions made by the largely compensating DeltaH and TDeltaS terms to the coupling free energy.     

Acyl carrier protein: structure-function relationships in a conserved multifunctional protein family.

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel alpha helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its "recognition" helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.     

ACTH1-17 is a more potent agonist at the human MC1 receptor than alpha-MSH.

The melanocortin receptor MC1 is expressed on melanocytes and is an important control point for melanogenesis and other responses. Alpha-MSH, which is considered to be the major ligand at the human melanocortin (MC)1 receptor (hMC1R), is produced from proopiomelanocortin (POMC) in the pituitary and in the skin by melanocytes and keratinocytes. Other POMC peptides are also produced in the skin and their concentrations exceed those of alpha-MSH by several fold. One of the most abundant is ACTH1-17. We have shown that adrenocorticotrophic hormone (ACTH)1-17 is more potent than alpha-MSH in stimulating melanogenesis in human melanocytes and unlike alpha-MSH produces a biphasic dose response curve. In this study we have examined the ability of ACTH1-17 to function as a ligand at the hMC1R. Competitive binding assays with [125I]Nle4 DPhe7 alpha-MSH as labelled ligand were carried out in HEK 293 cells transfected with the hMC1R. ACTH1-17 showed high affinity for the hMC1R with a Ki value of 0.21 +/- 0.03 nM which was slightly higher than that of 0.13 +/- 0.005 nM for alpha-MSH. ACTH1-17 was, however, more potent than alpha-MSH in increasing cAMP and IP3 production in the transfected cells. Our results demonstrate that ACTH1-17 is a potent agonist at the hMC1R. It is therefore possible that ACTH1-17, which is found in the skin in greater concentrations than alpha-MSH, has an important role in the regulation of human melanocytes and other cell types that express the hMC1R.     

Conformationally restricted nucleotides as a probe of structure-function relationships in RNA

We introduce the use of commercially available locked nucleic acids (LNAs) as a functional probe in RNA. LNA nucleotides contain a covalent linkage that restricts the pseudorotation phase of the ribose to C3'-endo (A-form). Introduction of an LNA at a single site thus allows the role of ribose structure and dynamics in RNA function to be assessed. We apply LNA probing at multiple sites to analyze self-cleavage in the lead-dependent ribozyme (leadzyme), thermodynamic stability in the UUCG tetraloop, and the kinetics of recognition of U1A protein by U1 snRNA hairpin II. In the leadzyme, locking a single guanosine residue into the C3'-endo pucker increases the catalytic rate by a factor of 20, despite the fact that X-ray crystallographic and NMR structures of the leadzyme ground state reported a C2'-endo conformation at this site. These results strongly suggest that a conformational change at this position is critical for catalytic function. Functional insights obtained in all three systems demonstrate the highly general applicability of LNA probing in analysis of the role of ribose orientation in RNA structure, dynamics, and function.     

Neuromorphic hardware databases for exploring structure-function relationships in the brain.

Neuromorphic hardware is the term used to describe full custom-designed integrated circuits, or silicon ''chips'', that are the product of neuromorphic engineering--a methodology for the synthesis of biologically inspired elements and systems, such as individual neurons, retinae, cochleas, oculomotor systems and central pattern generators. We focus on the implementation of neurons and networks of neurons, designed to illuminate structure-function relationships. Neuromorphic hardware can be constructed with either digital or analogue circuitry or with mixed-signal circuitry--a hybrid of the two. Currently, most examples of this type of hardware are constructed using analogue circuits, in complementary metal-oxide-semiconductor technology. The correspondence between these circuits and neurons, or networks of neurons, can exist at a number of levels. At the lowest level, this correspondence is between membrane ion channels and field-effect transistors. At higher levels, the correspondence is between whole conductances and firing behaviour, and filters and amplifiers, devices found in conventional integrated circuit design. Similarly, neuromorphic engineers can choose to design Hodgkin-Huxley model neurons, or reduced models, such as integrate-and-fire neurons. In addition to the choice of level, there is also choice within the design technique itself; for example, resistive and capacitive properties of the neuronal membrane can be constructed with extrinsic devices, or using the intrinsic properties of the materials from which the transistors themselves are composed. So, silicon neurons can be built, with dendritic, somatic and axonal structures, and endowed with ionic, synaptic and morphological properties. Examples of the structure-function relationships already explored using neuromorphic hardware include correlation detection and direction selectivity. Establishing a database for this hardware is valuable for two reasons: first, independently of neuroscientific motivations, the field of neuromorphic engineering would benefit greatly from a resource in which circuit designs could be stored in a form appropriate for reuse and re-fabrication. Analogue designers would benefit particularly from such a database, as there are no equivalents to the algorithmic design methods available to designers of digital circuits. Second, and more importantly for the purpose of this theme issue, is the possibility of a database of silicon neuron designs replicating specific neuronal types and morphologies. In the future, it may be possible to use an automated process to translate morphometric data directly into circuit design compatible formats. The question that needs to be addressed is: what could a neuromorphic hardware database contribute to the wider neuroscientific community that a conventional database could not? One answer is that neuromorphic hardware is expected to provide analogue sensory-motor systems for interfacing the computational power of symbolic, digital systems with the external, analogue environment. It is also expected to contribute to ongoing work in neural-silicon interfaces and prosthetics. Finally, there is a possibility that the use of evolving circuits, using reconfigurable hardware and genetic algorithms, will create an explosion in the number of designs available to the neuroscience community. All this creates the need for a database to be established, and it would be advantageous to set about this while the field is relatively young. This paper outlines a framework for the construction of a neuromorphic hardware database, for use in the biological exploration of structure-function relationships.     

Glucagon antagonists. Synthesis and inhibitory properties of Asp3-containing glucagon analogs

In an effort to find analogs of glucagon that would bind to the glucagon receptor of the rat liver membrane but would not activate membrane-bound adenyl cyclase, several hybrid molecules were synthesized which contained sequences from both glucagon and secretin. [Asp3, Glu9]Glucagon and [Asp3, Glu9, Arg12]glucagon were inactive in the adenyl cyclase assay even at high concentrations but retained some binding affinity for the receptor. They were able to displace 125I-glucagon completely from its receptor and could completely inhibit the activation of adenyl cyclase by natural or synthetic glucagon. The inhibition index [I/A]50 was approximately 110 for both analogs. [Asp3]Glucagon, [Glu3]glucagon and [Asp3, Lys17, 18, Glu21]glucagon were weak partial agonists, while [Asp3, Glu21]glucagon was inactive and a poor inhibitor. The peptides were synthesized by solid-phase methods and purified to homogeneity by reverse-phase high-performance liquid chromatography on C18 silica columns. These are the first fully synthetic competitive glucagon antagonists to be reported.     

AMP is an adenosine A1 receptor agonist

Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5'-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5'-monophosphonate, ACP) directly activated the adenosine A(1) receptor (A(1)R). In contrast, AMP only activated the adenosine A(2B) receptor (A(2B)R) after hydrolysis to adenosine by ecto-5'-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A(1)R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A(1)R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A(1)R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A(1)R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine.     

Two subforms of eukaryotic topoisomerase I. Purification and structure-function relationships.

A new method for isolation of eukaryotic topoisomerase I from calf thymus and from Jurkat-1 cells using HPLC has been developed. The method allows quantitative purification of high molecular weight topo I and two low molecular weight fractions differing by their isoelectric points. It has been suggested that these fractions be characterized as two subforms of the enzyme possessing structural and functional differences. The differences in their specific activities, sensitivity to camptothecin and in their proteolytic digestion maps have been demonstrated for the two enzymes.     

Insulin and glucagon relationships during aging in rats.

Oral glucose tolerance tests were performed under pentobarbital anesthesia in 43 male Wistar rats 2 to 18 months of age in order to determine if insulin and glucagon secretion are altered with aging. Although any linear correlation was not demonstrated between aging and blood glucose, plasma insulin or glucagon levels, post-glucose levels of blood glucose were significantly suppressed and those of plasma glucagon were significantly elevated at 4 to 6 months of age. No significant difference was found between young (2 months of age) and aged rats (12 to 14 and 17 to 18 months of age) in either blood glucose or plasma insulin levels during oral glucose load. On the other hand, post-glucose plasma glucagon levels of the aged rats were significantly higher than those of the young ones. Furthermore, comparisons of various kinds of indices among the different age groups, such as insulinogenic index, insulin/glucagon and so forth during oral glucose tolerance tests also indicate the significant alteration of glucagon secretion during aging process. It is concluded from the present data that glucose tolerance does not apparently deteriorate during aging process in rats but that glucagon responses to oral glucose administration are elevated with aging.     

Augusta disease in tulip - a reassessment

In an experiment in which the roots of field-grown tulip were commonly infected with tobacco necrosis virus (TNV), Augusta disease did not develop in the year of infection or when progeny bulbs were grown in the field or glass-house. When tulip bulbs of other stocks, including grades of 11 and 12 cm circumference, were forced, the disease developed sporadically, in some instances as the result of infection with TNV from the soil in which they were planted and in others as a result of infection by bulb-borne virus. The incidence of disease produced by current year infection was increased by warming the plunge bed. Different strains of TNV were obtained from field-grown plants with Augusta disease and different strains of the virus produced the disease when inoculated to tulip. Some, but not all, naturally diseased plants contained satellite virus, which therefore does not cause or prevent disease development. The disease was produced in some plants by TNV transmitted by Olpidium brassicae, but neither a vector nor a non-vector isolate of O. brassicae completed its life cycle in tulip. However, Olpidium-like zoospores were observed in some washings of tulip roots from TNV-infested soils. TNV was not obtained from all tulip plants with necrotic leaf symptoms resembling Augusta disease. Some were infected with tomato bushy stunt virus or cucumber mosaic virus, or with another agent that was transmitted by inoculation of sap to Nicotiana clevelandii and Chenopodium quinoa, and carried by bulbs of up to 11 cm circumference.     

Different structure-function relationships for alpha-factor-induced morphogenesis and agglutination in Saccharomyces cerevisiae.

Eight synthetic analogs of the mating pheromone alpha-factor-induced morphogenesis and increased agglutinability in a cells. Most analogs induced increased agglutinability at lower concentrations than those at which they induced morphogenesis, but the ratio of the potencies for the two effects varied 140-fold among different analogs. Morphological response to pheromone required exposure for at least 90 min, but increased agglutinability followed exposures of 20 s. Two synthetic analogs induced neither response. In competition experiments, both of these analogs prevented induction of increased agglutinability and morphogenesis by active alpha factor. The inactive peptides blocked increased agglutinability at lower concentrations than those at which they blocked morphogenesis. alpha factors exhibited different structure-function relationships for morphogenesis as compared with agglutinability. Thus, response of Saccharomyces cerevisiae to alpha factor is complex and may be mediated by more than one receptor.