Ice-binding proteins adsorb to their ligand via anchored clathrate waters

The main success of my thesis has been to establish the mechanism by which antifreeze proteins (AFPs) bind irreversibly to ice crystals, and hence prevent their growth. AFPs organize ice-like water on their ice-binding site, which then merges and freezes with the quasi-liquid layer of ice. This was revealed from studying the exceptionally large (ca. 1.5-MDa) Ca 2+-dependent AFP from the Antarctic bacterium Marinomonas primoryensis (MpAFP). The 34-kDa antifreeze- active region of MpAFP was predicted to fold as a novel Ca 2+-binding β-helix. Site-directed mutagenesis confirmed the model and demonstrated that its ice-binding site (IBS) consisted of solvent-exposed Thr and Asx parallel arrays on the Ca 2+-binding turns. The X-ray crystal structure of the antifreeze region was solved to a resolution of 1.7 Å. Two of the four molecules within the unit cell of the crystal had portions of their IBSs freely exposed to solvent. Identical clathrate-like cages of water molecules were present on each IBS. These waters were organized by the hydrophobic effect and anchored to the protein via hydrogen bonds. They matched the spacing of water molecules in an ice lattice, demonstrating that anchored clathrate waters bind AFPs to ice. This mechanism was extended to other AFPs including the globular type III AFP from fishes. Site-directed mutagenesis and a modified ice-etching technique demonstrated this protein uses a compound ice-binding site, comprised of two flat and relatively hydrophobic surfaces, to bind at least two planes of ice. Reinvestigation of several crystal structures of type III AFP identified anchored clathrate waters on the solvent-exposed portion of its compound IBS that matched the spacing of waters on the primary prism plane of ice. Ice nucleation proteins (INPs), which can raise the temperature at which ice forms in solution to just slightly below 0oC, have the opposite effect to AFPs. A novel dimeric β-helical model was proposed for the INP produced by the bacterium Pseudomonas borealis. Molecular dynamics simulations showed that INPs are also capable of ordering water molecules into an ice- like lattice. However, their multimerization brings together sufficient ordered waters to form an ice nucleus and initiate freezing.

Apoptosis and cell-cycle regulatory proteins in colorectal carcinoma: relationship to tumour stage and patient survival

The quantitative assessment of apoptotic index (AI) and mitotic index (MI) and the immunoreactivity of p53, bcl-2, p21, and mdm2 were examined in tumour and adjacent normal tissue samples from 30 patients with colonic and 22 with rectal adenocarcinoma. Individual features and combined profiles were correlated with clinicopathological parameters and patient survival data to assess their prognostic value. Increased AI was significantly associated with increased bcl-2 expression (p

Positive and negative contractile effects of somatostatin-14 on rat ventricular cardiomyocytes.

Somatostatin-14 elicits negative inotropic and chronotropic actions in atrial myocardium. Less is known about the effects of somatostatin-14 in ventricular myocardium. The direct contractile effects of somatostatin-14 were assessed using ventricular cardiomyocytes isolated from the hearts of adult rats. Cells were stimulated at 0.5 Hz with CaCl2 (2 mM) under basal conditions and in the presence of the -adrenoceptor agonist, isoprenaline (1 nM), or the selective inhibitor of the transient outward current (Ito), 4-aminopyridine (500 M). Somatostatin-14 did not alter basal contractile response but it did inhibit (IC50 13 nM) the response to isoprenaline (1 nM). In the presence of 4-aminopyridine (500 M), somatostatin-14 stimulated a positive contractile response (EC50 118 fM) that was attenuated markedly by diltiazem (100 nM). These data indicate that somatostatin-14 exerts dual effects directly in rat ventricular cardiomyocytes: (1) a negative contractile effect, observed in the presence of isoprenaline (1 nM), coupled to activation of Ito; and (2) a previously unreported and very potent positive contractile effect, unmasked by 4-aminopyridine (500 M), coupled to the influx of calcium ions via L-type calcium channels. The greater potency of somatostatin-14 for producing the positive contractile effect indicates that the peptide may exert a predominantly stimulatory influence on the resting contractility of ventricular myocardium in vivo, whereas the negative contractile effect, observed at much higher concentrations, could indicate that localized elevations in the concentration of the peptide may serve as a negative regulatory influence to limit the detrimental effects of excessive stimulation of cardiomyocyte contractility.

Modulation of contractile function through NPY receptors during development of cardiomyocyte hypertrophy.

Severity of left ventricular hypertrophy (LVH) correlates with elevated plasma levels of neuropeptide Y (NPY) in hypertension. NPY elicits positive and negative contractile effects in cardiomyocytes through Y(1) and Y(2) receptors, respectively. This study tested the hypothesis that NPY receptor-mediated contraction is altered during progression of LVH. Ventricular cardiomyocytes were isolated from spontaneously hypertensive rats (SHRs) pre-LVH (12 weeks), during development (16 weeks), and at established LVH (20 weeks) and age-matched normotensive Wistar Kyoto (WKY) rats. Electrically stimulated (60 V, 0.5 Hz) cell shortening was measured using edge detection and receptor expression determined at mRNA and protein level. The NPY and Y(1) receptor-selective agonist, Leu(31)Pro(34)NPY, stimulated increases in contractile amplitude, which were abolished by the Y(1) receptor-selective antagonist, BIBP3226 [R-N(2)-(diphenyl-acetyl)-N-(4-hydroxyphenyl)methyl-argininamide)], confirming Y(1) receptor involvement. Potencies of both agonists were enhanced in SHR cardiomyocytes at 20 weeks (2300- and 380-fold versus controls). Maximal responses were not attenuated. BIBP3226 unmasked a negative contraction effect of NPY, elicited over the concentration range (10(-12) to 3 x 10(-9) M) in which NPY and PYY(3-36) attenuated the positive contraction effects of isoproterenol, the potencies of which were increased in cardiomyocytes from SHRs at 20 weeks (175- and 145-fold versus controls); maximal responses were not altered. Expression of NPY-Y(1) and NPY-Y(2) receptor mRNAs was decreased (55 and 69%) in left ventricular cardiomyocytes from 20-week-old SHRs versus age-matched WKY rats; parallel decreases (32 and 80%) were observed at protein level. Enhancement of NPY potency, producing (opposing) contractile effects on cardiomyocytes together with unchanged maximal response despite reduced receptor number, enables NPY to contribute to regulating cardiac performance during compensatory LVH.

Characterisation of two calmodulin-like proteins from the liver fluke, Fasciola hepatica.

Calmodulin is a calcium ion-sensing signalling protein found in eukaryotics. Two calmodulin-like gene sequences were identified in an EST library from adult liver flukes. One codes for a protein (FhCaM1) homologous to mammalian calmodulins (98% identity), whereas the other protein (FhCaM2) has only 41% identity. These genes were cloned into expression vectors and the recombinant proteins were expressed in Escherichia coli. Gel shift assays showed that both proteins bind to calcium, magnesium and zinc ions. Homology models have been built for both proteins. As expected, FhCaM1 has a highly similar structure to other calmodulins. Although FhCaM2 has a similar fold, its surface charge is higher than FhCaM1. One of the potential metal ion-binding sites has lower metal-ion co-ordination capability, while another has an adjacent lysine residue, both of which may decrease the metal-binding affinity. These differences may reflect a specialised role for FhCaM2 in the liver fluke.