Validated ligand binding sites in CCK receptors. Next step: Computer aided design of novel CCK ligands

Computer-aided drug design becomes an important part of G-protein coupled receptors (GPCR) drug discovery process that is applied for improving the efficiency of derivation and optimization of novel ligands. It represents the combination of methods that-use-structural information of a receptor binding site of known ligands to design new ligands. In this report, we give a brief description of ligand binding sites in cholecystokinin and gastrin receptors (CK1R and CCK2R) which were delineated using experimental and computational methods, and then, we show how the validated ligand binding sites can be used to design and improve novel ligands. The translation of the knowledge of ligand-binding sites of different GPCRs to computer-aided design of novel ligands is summarized.

Modeled structure of a G-protein-coupled receptor:The cholecystokinin-1 receptor

The Cholecystokinin-1 receptor (CCK1R) mediates actions of CCK in areas of the central nervous system and of the gut. It is a potential target to treat a number of diseases. As for all G-protein-coupled receptors, docking of ligands into modeled CCK1R binding site should greatly help to understand intrinsic mechanisms of activation. Here, we describe the procedure we used to progressively build a structural model for the CCK1R, to integrated, and on the basis of site-directed mutagenesis data on its binding site. Reliability of the CCK1R model was confirmed by interaction networks that involved conserved and functionally crucial motifs in G-protein-coupled receptors, such as Glu/Asp-Arg-Tyr and Asn-Pro-Xaa-Xaa-Tyr motifs. In addition, the 3-D structure of CCK1R-bound CCK resembled that determined by NMR in a lipid environment. The derived computational model was also used for revealing binding modes of several nonpeptide ligands and for rationalizing ligand structure-activity relationships known from experiments. Our findings indeed support that our "validated CCK1R model" could be used to study the intrinsic mechanism of CCK1R activation and design new ligands.

Coelum non animum muto? Milton's Neo-Latin Poetry and Catholic Italy

Milton’s Elegiarum Liber, the first half of his Poemata published in Poems of Mr John Milton Both English and Latin (1645), concludes with a series of eight Latin epigrams: five bitterly anti-Catholic pieces on the failed Gunpowder Plot of 1605, followed by three encomiastic poems hymning the praises of an Italian soprano, Leonora Baroni, singing in Catholic Rome. The disparity in terms of subject matter and tone is self-evident yet surprising in an epigrammatic series that runs sequentially. Whereas the gunpowder epigrams denigrate Rome, the Leonora epigrams present the city as a cultured hub of inclusivity, the welcome host of a Neapolitan soprano. In providing the setting for a human song that both enthrals its audience and attests to the presence of a divine power, Rome now epitomizes something other than brute idolatry, clerical habit or doctrine. And for the poet this facilitates an interrogation of theological (especially Catholic) doctrines. Coelum non animum muto, dum trans mare curro wrote the homeward-bound Milton in the autograph book of Camillo Cardoini at Geneva on 10 June 1639. But that this was an animus that could indeed acclimatize to religious and cultural difference is suggested by the Latin poems which Milton “patch [ed] up” in the course of his Italian journey. Central to that acclimatisation, as this chapter argues, is Milton’s quasi-Catholic self-fashioning. Thus Mansus offers a poetic autobiography of sorts, a self-inscribed vita coloured by intertextually kaleidoscopic links with two Catholic poets of Renaissance Italy and their patron; Ad Leonoram 1 both invokes and interrogates Catholic doctrine before a Catholic audience only to view the whole through the lens of a neo-Platonic hermeticism that may refreshingly transcend religious difference. Finally, Epitaphium Damonis, composed upon Milton’s return home, seems to highlight the potential interconnectedness of Protestant England and Catholic Italy, through the Anglo-Italian identity of its deceased subject, and through a pseudo-monasticism suggested by the poem���s possible engagement with the hagiography of a Catholic Saint. Perhaps continental travel and the physical encounter with the symbols, personages and institutions of the other have engendered in the Milton of the Italian journey a tolerance or, more accurately, the manipulation of a seeming tolerance to serve poetic and cultural ends.


First reviewer:
Haan: a fine piece by the senior neo-Latinist in Milton studies.

Second reviewer:
Chapter 7 is ... a high-spot of the collection. Its argument that in his Latin poetry Milton’s is a ‘quasi-Catholic self-fashioning’ stressing ‘the potential interconnectedness of Protestant England and Catholic Italy’ is striking and is advanced with learning, clarity and insight. Its sensitive exploration of the paradox of Milton’s coupling of humanistically complimentary and tolerant address to Roman Catholic friends with fiercely Protestant partisanship demonstrates that there is much greater complexity to his poetic persona than the self-construction and self-presentation of the later works would suggest. The essay is always adroit and sure-footed, often critically acute and illuminating (as, for example, in its discussion of the adjective and adverb mollis and molliter in Mansus, or in the identification in n. 99 of hitherto unnoticed Virgilian echoes). It has the added merits of being very well written, precise and apt in its citation of evidence, and absolutely central to the concerns of the volume.

Treatment of age-related subfoveal neovascular membranes by teletherapy:a pilot study

This investigation was designed to determine whether low dose radiation to the macular region could influence the natural course of age-related subfoveal neovascularisation. Nineteen patients with subfoveal membranes due to age-related macular degeneration (ARMD) were treated with 10 or 15 Gy of 6 MV photons and seven patients who declined treatment were followed up as controls. Six controls and all treated patients had completed follow up times of at least 12 months. Visual acuity was maintained or improved in 78% and 63% of treated patients at their 6 and 12 month follow up examinations respectively. By contrast visual acuity showed steady deterioration in six of seven controls. Significant neovascular membrane regression, as measured by image analysis, was recorded in 68% and 77% of treated patients at 6 and 12 months post-radiation, whereas the membranes in all seven control patients showed progressive enlargement. This study suggests that low doses of radiation can maintain central vision and induce regression of subfoveal neovascular membranes of ARMD in a significant proportion of patients. We now believe it appropriate to proceed to a prospective randomised study to test this hypothesis further.

'Latinising' Milton: Paradise Lost, Latinitas and the Long Eighteenth Century

This chapter traces the trajectory of Latin translations of Milton’s vernacular verse most capably encapsulated by Latin verse paraphrases of Paradise Lost by a certain J.C. (1686), William Hog (1690), Thomas Power (1691) and by such eighteenth-century renderings as that of William Dobson (1753). Situating its analysis in relation to early modern pedagogical practices, including the double translation system, and informed by current translational theory, the analysis considers the multifunctional aims and consequences of Latinising Milton: the elaboration and elucidation of a vernacular original via Latin exegesis and paraphrase; recourse to Latin as a means of facilitating a wider European readership. Integral to the discussion is an alertness to the contemporary and later reception of Milton’s work, and an assessment of ways in which Latinitas enabled the invocation of classical intertexts which in themselves offer a nuanced reading of Miltonic verse.

'Translated Verse': Milton's Latin Poetry in the Long Eighteenth Century

This chapter assesses ways in which the emergence in the long eighteenth century of a cluster of verse translations of Milton’s Poemata engendered an intellectual discourse and debate on translation itself, not dissimilar to the magazine warfare of the day. It argues that poetical renderings of Milton’s Latin verse, and the biographical and literary contexts in which they appeared, facilitated the interrogation of key issues that are still being debated by modern translation theorists: the nature and function of translation; the viability of rendering a source text in a target language that is also in this instance a poetic language; the potential ‘fetters’ which, in Drydenesque terms, might constrain ‘the verbal copier’; or by contrast the quasi-liberating fluency, the ‘fluent strategy’, attendant upon recourse to verse as translational medium; canonicity, amplification and omission; the much-debated issue of authorial equivalence, evinced here, it is suggested, by the editorial showcasing of the translator; and not least, the perennial question of translation as reading and critical interpretation. In short, verse renderings of Milton’s Latin poetry and the debates that they engendered assume a not inconsequential place in the history of translation theory, which, as Venuti notes, is forever concerned with ‘the changing relationships between the relative autonomy of the translated text and two other categories: equivalence and function.’

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Nerve pathology in the type 1 diabetic dog: effects of treatment with sulindac

The purpose of this study was to define pathological abnormalities in the peripheral nerve of a large animal model of long-duration type 1 diabetes and also to determine the effects of treatment with sulindac. Detailed morphometric studies were performed to define nerve fiber and endoneurial capillary pathology in 6 control dogs, 6 type 1 diabetic dogs treated with insulin, and 6 type 1 diabetic dogs treated with insulin and sulindac for 4 years. Myelinated fiber and regenerative cluster density showed a non-significant trend toward a reduction in diabetic compared to control animals, which was prevented by treatment with sulindac. Unmyelinated fiber density did not differ among groups. However, diabetic animals showed a non-significant trend toward an increase in axon diameter (p <0.07), with a shift of the size frequency distribution towards larger axons, which was not prevented by treatment with sulindac. Endoneurial capillary density and luminal area showed a non-significant trend toward an increase in diabetic animals, which was prevented with sulindac treatment. Endoneurial capillary basement membrane area was significantly increased (p <0.05) in diabetic animals, but was not prevented with sulindac treatment. We conclude that the type 1 diabetic dog demonstrates minor structural abnormalities in the nerve fibers and endoneurial capillaries of the sciatic nerve, and treatment with sulindac ameliorates some but not all of these abnormalities.

Diabetic retinopathy: morphometric analysis of basement membrane thickening of capillaries in different retinal layers within arterial and venous environments

AIMS: To assess quantitatively variations in the extent of capillary basement membrane (BM) thickening between different retinal layers and within arterial and venous environments during diabetes. METHODS: One year after induction of experimental (streptozotocin) diabetes in rats, six diabetic animals together with six age-matched control animals were sacrificed and the retinas fixed for transmission electron microscopy (TEM). Blocks of retina straddling the major arteries and veins in the central retinal were dissected out, embedded in resin, and sectioned. Capillaries in close proximity to arteries or veins were designated as residing in either an arterial (AE) or a venous (VE) environment respectively, and the retinal layer in which each capillary was located was also noted. The thickness of the BM was then measured on an image analyser based two dimensional morphometric analysis system. RESULTS: In both diabetics and controls the AE capillaries had consistently thicker BMs than the VE capillaries. The BMs of both AE and VE capillaries from diabetics were thicker than those of capillaries in the corresponding retinal layer from the normal rats (p <or = 0.005). Also, in normal AE and VE capillaries and diabetic AE capillaries the BM in the nerve fibre layer (NFL) was thicker than that in either the inner (IPL) or outer (OPL) plexiform layers (p <or = 0.001). However, in diabetic VE capillaries the BMs of capillaries in the NFL were thicker than those of capillaries in the IPL (p <or = 0.05) which, in turn, had thicker BMs than capillaries in the OPL (p <or = 0.005). CONCLUSIONS: The variation in the extent of capillary BM thickening between different retinal layers within AE and VE environments may be related to differences in levels of oxygen tension and oxidative stress in the retina around arteries compared with that around veins.

Morphometric analysis of corneal endothelial giant cells in normal and traumatized corneas

Corneal endothelial cells from normal and traumatized human, primate, cat and rabbit eyes were studied by specular microscopy. Morphometric analysis was performed on micrographs of corneal endothelium using a semi-automated image analysis system. The results showed that under normal conditions the corneal endothelium of all four species exhibit major morphological similarities (mean cell areas: human 317 +/- 32 microns 2, primate 246 +/- 22 microns2, cat 357 +/- 25 microns 2, rabbit 308 +/- 35 microns 2). The normal corneal endothelium in man was found to be more polymegethous than that of the other species. Trauma to cat, primate and human corneas resulted in a long-term reduction in endothelial cell density and enhanced polymegethism. In contrast, the reparative response of the rabbit ensured the reformation of an essentially normal monolayer following injury. Endothelial giant cells were a normal inclusion in the rabbit corneal endothelium but were only significant in cat, primate and man following trauma. The presence of corneal endothelial giant cells in amitotic corneas may therefore represent a compensatory response in the absence of mitotic potential.

Retinal and choroidal responses to panretinal photocoagulation: an ultrastructural perspective

BACKGROUND: There have been few histological or ultrastructural studies of the outer retina and choriocapillaris following panretinal photocoagulation therapy. This investigation examines the long-term morphological effects of panretinal photocoagulation in two patients with type II diabetes who had received laser treatment more than 6 months prior to death.

METHODS: Regions of retina and choroid from each patient were fixed in 2.5% glutaraldehyde, dissected out and examined using light microscopy and scanning and transmission electron microscopy.

RESULTS: After removing the neural retina, scanning electron microscopy of non-photocoagulated areas of the eye cups revealed normal cobblestone-like retinal pigment epithelial (RPE) cells. Regions with laser scars showed little RPE infiltration into the scar area, although large rounded cells often appeared in isolation within these areas. Sections of the retina and choroid in burn regions showed a complete absence of the outer nuclear layer and photoreceptor cells, with the inner retinal layers lying in close apposition to Bruch's membrane. Non-photocoagulated regions of the retina and choroid appeared normal in terms of both cell number and cell distribution. The RPE layer was absent within burn scars but many RPE-like cells appeared markedly hypertrophic at the edges of these regions. Bruch's membrane always remained intact, although the underlying choriocapillaris was clearly disrupted at the point of photocoagulation burns, appearing largely fibrosed and non-perfused. Occasional choroidal capillaries occurring in this region were typically small in profile and had plump non-fenestrated endothelium.

CONCLUSIONS: This study outlines retinal and choroidal cell responses to panretinal photocoagulation in diabetic patients and demonstrates an apparent reduction in the capacity of these tissues to repair laser damage.

Stereological estimation of Weibel-Palade bodies in the retinal vasculature of normal and diabetic dogs

The absolute volume of Weibel-Palade (WP) bodies, the storage organelles of von Willebrand factor (vWF), was estimated by a stereological method in a known volume of central retina from normal and 5-year diabetic dogs. The results showed that the volume of WP bodies present in the endothelium of the retinal vasculature varies with blood vessel type and in diabetes. In both diabetic and normal dogs the endothelium of the retinal veins contained a higher volume of WP bodies than that of the retinal arteries. In dogs which had been diabetic for a duration of 5 years the volume of WP bodies present in the endothelium of retinal veins was significantly greater than in the endothelium of veins from the control animals. However, there was no significant difference in the volume of WP bodies present in the endothelium of retinal arteries or capillaries between the two groups of animals.

Estimation of the surface area and volume of the retinal capillary basement membrane using the stereologic method of vertical sections

The modern stereologic method of vertical sections was applied to the retina as a means of generating unbiased estimates of three-dimensional structure. The method is illustrated with real data on the volume and surface area of the capillary basement membrane from the central retina of the rat. Novel methods of estimating the volume of retina sampled and of creating accurate vertical sections are described. The advantages of using stereologic methods to generate quantitative information on the three-dimensional structure of the retina are discussed and compared to those of previous quantitative methods that provide data on two-dimensional structure only.

Ionizing radiation and the retina

This review considers the effects of ionizing radiation on the retina and examines the relationship between the natural course of radiation retinopathy and the radiobiology of the retinal vascular endothelial cell (RVEC). Radiation retinopathy presents clinically as a progressive pattern of degenerative and proliferative vascular changes, chiefly affecting the macula, and ranging from capillary occlusion, dilation, and microaneurysm formation, to telangiectasia, intraretinal microvascular abnormalities, and neovascularization. The total-radiation dose and fractionation schedule are the major determinants for the time of onset, rate of progression, and severity of retinopathy, although other factors such as concomitant chemotherapy and preexisting diabetes may exaggerate the vasculopathy by intensifying the oxygen-derived free-radical assault on the vascular cells. The differential radiosensitivity of RVECs is attributed to their nuclear chromatin conformation, their antioxidant status, and their environment. We propose pathogenetic mechanisms for radiation retinopathy and suggest that the peculiar latency and unique clinical pattern is related to the life cycle of the RVEC. A rationale is also proposed for the use of radiotherapy in the treatment of subneovascularization and age-related macular degeneration.