Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding.

Insulin-like growth factor-1 (IGF-1) is a serum protein which unexpectedly folds to yield two stable tertiary structures with different disulphide connectivities; native IGF-1 [18-61,6-48,47-52] and IGF-1 swap [18-61,6-47, 48-52]. Here we demonstrate in detail the biological properties of recombinant human native IGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae. IGF-1 swap had a approximately 30 fold loss in affinity for the IGF-1 receptor overexpressed on BHK cells compared with native IGF-1.The parallel increase in dose required to induce negative cooperativity together with the parallel loss in mitogenicity in NIH 3T3 cells implies that disruption of the IGF-1 receptor binding interaction rather than restriction of a post-binding conformational change is responsible for the reduction in biological activity of IGF-1 swap. Interestingly, the affinity of IGF-1 swap for the insulin receptor was approximately 200 fold lower than that of native IGF-1 indicating that the binding surface complementary to the insulin receptor (or the ability to attain it) is disturbed to a greater extent than that to the IGF-1 receptor. A 1.0 ns high-temperature molecular dynamics study of the local energy landscape of IGF-1 swap resulted in uncoiling of the first A-region alpha-helix and a rearrangement in the relative orientation of the A- and B-regions. The model of IGF-1 swap is structurally homologous to the NMR structure of insulin swap and CD spectra consistent with the model are presented. However, in the model of IGF-1 swap the C-region has filled the space where the first A-region alpha-helix has uncoiled and this may be hindering interaction of Val44 with the second insulin receptor binding pocket.     

Centrally Administered Ghrelin Acutely Influences Food Choice in Rodents

We sought to determine whether the orexigenic hormone, ghrelin, is involved in the intrinsic regulation of food choice in rats. Ghrelin would seem suited to serve such a role given that it signals hunger information from the stomach to brain areas important for feeding control, including the hypothalamus and reward system (e.g. ventral tegmental area, VTA). Thus, in rats offered a choice of palatable foods (sucrose pellets and lard) superimposed on regular chow for 2 weeks, we explored whether acute central delivery of ghrelin (intracerebroventricular (ICV) or intra-VTA) is able to redirect their dietary choice. The major unexpected finding is that, in rats with high baseline lard intake, acute ICV ghrelin injection increased their chow intake over 3-fold, relative to vehicle-injected controls, measured at both 3 hr and 6 hr after injection. Similar effects were observed when ghrelin was delivered to the VTA, thereby identifying the VTA as a likely contributing neurobiological substrate for these effects. We also explored food choice after an overnight fast, when endogenous ghrelin levels are elevated, and found similar effects of dietary choice to those described for ghrelin. These effects of fasting on food choice were suppressed in models of suppressed ghrelin signaling (i.e. peripheral injection of a ghrelin receptor antagonist to rats and ghrelin receptor (GHSR) knock-out mice), implicating a role for endogenous ghrelin in the changes in food choice that occur after an overnight fast. Thus, in line with its role as a gut-brain hunger hormone, ghrelin appears to be able to acutely alter food choice, with notable effects to promote “healthy” chow intake, and identify the VTA as a likely contributing neurobiological substrate for these effects.     

Cardiac Fibroblasts Contribute to Myocardial Dysfunction in Mice with Sepsis: The Role of NLRP3 Inflammasome Activation

Myocardial contractile dysfunction in sepsis is associated with the increased morbidity and mortality. Although the underlying mechanisms of the cardiac depression have not been fully elucidated, an exaggerated inflammatory response is believed to be responsible. Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome is an intracellular platform that is involved in the maturation and release of interleukin (IL)-1β. The aim of the present study is to evaluate whether sepsis activates NLRP3 inflammasome/caspase-1/IL-1β pathway in cardiac fibroblasts (CFs) and whether this cytokine can subsequently impact the function of cardiomyocytes (cardiac fibroblast-myocyte cross-talk). We show that treatment of CFs with lipopolysaccharide (LPS) induces upregulation of NLRP3, activation of caspase-1, as well as the maturation (activation) and release of IL-1β. In addition, the genetic (small interfering ribonucleic acid [siRNA]) and pharmacological (glyburide) inhibition of the NLRP3 inflammasome in CFs can block this signaling pathway. Furthermore, the inhibition of the NLRP3 inflammasome in cardiac fibroblasts ameliorated the ability of LPS-chalenged CFs to impact cardiomyocyte function as assessed by intracellular cyclic adenosine monophosphate (cAMP) responses in cardiomyocytes. Salient features of this the NLP3 inflammasome/ caspase-1 pathway were confirmed in in vivo models of endotoxemia/sepsis. We found that inhibition of the NLRP3 inflammasome attenuated myocardial dysfunction in mice with LPS and increased the survival rate in mice with feces-induced peritonitis. Our results indicate that the activation of the NLRP3 inflammasome in cardiac fibroblasts is pivotal in the induction of myocardial dysfunction in sepsis.     

Expression of mosquitocidal crystal protein genes in non-insecticidal Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis NTB-1 isolated from soil samples in Korea produces ovoidal parasporal inclusions with proteins of approximately 24–40 kDa in size. Although serological study indicated that the isolate has a flagella (H) antigen identical with subsp. israelensis , it seemed to be non-insecticidal against Lepidoptera and Coleoptera as well as Diptera. To investigate the activity of non-insecticidal B. thuringiensis transformed with insecticidal crystal protein genes, cryIVD and cytA genes of B. thuringiensis subsp. morrisoni PG-14, highly toxic to mosquito larvae, were introduced into the isolate NTB-1. The expression of mosquitocidal crystal protein genes in NTB-1 was characterized by SDS–PAGE analysis and electron microscopy. The results showed that crystalline inclusions of host, CryIVD and CytA were stably expressed in the transformant. However, the mosquitocidal activity of transformant was similar to that of B. thuringiensis subsp. kurstaki Cry⁻ B harbouring cryIVD and cytA genes, demonstrating that a synergistic effect by an interaction of both introduced insecticidal and resident non-insecticidal crystal proteins was not observed.     

Aerobic photolysis of alkylcobalamins: quantum yields and light-action spectra

Quantum yields (φ) for the aerobic photolysis of 5′-deoxyadenosylcobalamin (dAB₁₂), methylcobalamin (MeB₁₂), propylcobalamin (PrB₁₂), and ethylcobalamin (EtB₁₂) were determined as a function of the irradiation wavelength. φ Determinations were made for both the base-on and base-off forms of each compound (except base-off dAB₁₂) at incident wavelengths from 250 nm to 570 nm. As a rule, the φs were high (0.1–0.5) and they varied significantly with respect to the irradiation wavelength. In general, each alkylcobalamin at pH 7.0 displayed a quantum yield spectrum distinct from its base-off form at pH 1.0. Across most of the spectrum, the φs of the base-off form were appreciably smaller than the base-on φs of the same compound. An exception to this generality was MeB₁₂ for which the φs at pH 1.0 were about the same as, or slightly greater above 450 nm than those at pH 7.0. At pH 7.0 and in the visible region the trend of the φs was dAB₁₂ < MeB₁₂ < PrB₁₂ < EtB₁₂. Under neutral conditions each compound showed a broad quantum yield peak in the 450–470 nm region.From the quantum yield and absorption spectra, photolysis spectra were calculated for 5.0 × 10^?5m solutions of each compound. The light-action spectra accurately give the relative rates/μ Einstein that these solutions photolyze at each wavelength. Thus, for example, MeB₁₂ photolyzed faster at pH 7.0 versus pH 1.0 in 510 nm light, but it photolyzed slower at pH 7.0 versus pH 1.0 in 450 nm light. Solutions of each compound photolyzed faster in the ultraviolet region as opposed to the visible (e.g., 310 nm versus 510 nm).Our findings show that the previously reported photolysis rates estimated by others with tungsten lamps provide no valid information about the intrinsic photolability of various alkyl-cobalt bonds. This also applies to the relative white-light photolysis rates reported for the base-on versus the base-off form of MeB₁₂. All such relative rates are artifacts which represent only the extent of overlap between the true action spectrum and the light emission spectrum of an incandescent lamp.     

Generalized binding phenomena in an allosteric macromolecule

A general macromolecular partition function is developed in terms of chemical ligand activity, temperature and pressure for systems described by an array of species which are characterized by their state of allosteric conformation and ligand stoichiometry. The effects of chemical ligand binding, enthalpy change, and volume change are treated in a parallel manner. From a broad viewpoint all of these effects can be regarded as specific cases of generalized binding phenomena. This approach provides a general method for analyzing calorimetric and ligand binding experiments. Several applications are given: (1) Thermal scanning data for tRNAphe (P.L. Privalov and V.V. Filimonov, J. Mol. Biol. 122 (1978) 447) are shown to fit a general model with six conformational states. By application of linkage theory it is shown that sodium chloride is expelled as the molecule denatures. (2) The results of calorimetric titrations on the arabinose binding protein (H. Fukada, J.M. Sturtevant and F.A. Quiocho, J. Mol. Biol. 258 (1983) 13193) are shown to fit a simple two-state allosteric model. (3) A thermal binding curve is simulated for an unusual respiratory protein, trout I hemoglobin (B.G. Barisas and S.J. Gill, Biophys. Chem. 9 (1979) 235), in order to illustrate both the similarities and differences between enthalpy and chemical ligand binding processes.