Eruptive lentigines confined to psoriatic plaques following biologic therapy

Clinical Rheumatology -     

Carcinoid tumours of the larynx

The larynx is a rare site of origin of neuroendocrine carcinomas. They can be divided into typical, atypical and small cell tumours on the basis of their histopathological differentiation. The tumour histology and prognosis correlate closely. The typical carcinoid tumours are well differentiated with a benign course. Conservative surgery for local disease is the treatment and is associated with good survival. The atypical carcinoid tumours are poorly differentiated with an aggressive course. Response to radiotherapy and chemotherapy is poor. The treatment of choice is adequate total excision of the lesion with neck dissection if there is clinical evidence of cervical lymphadenopathy and a careful follow-up so as to recognise and treat any metastatic spread. We present two cases with similar history and clinical findings—one typical and the other atypical—and discuss the relevant literature.     

Induction of cortical cataracts in cultured mouse lenses with H-89, an inhibitor of protein kinase A

PURPOSE: To compare the effects of two serine-threonine protein kinase inhibitors in a mouse lens culture system previously designed to investigate cortical cataracts caused by L-buthionine sulfoximine (BSO), inhibitor of GSH biosynthesis. METHODS: Cataract development in HL-1 medium was evaluated visually or by measurement of lens Na+/K+ ratio through atomic absorption. Protein changes were evaluated by 32P-labeling, 2D-gel electrophoresis, phosphorimaging and mass spectrometry. Results. H-7 (50 microM), inhibitor of protein kinase A (PKA) and protein kinase C (PKC), did not cause cataracts, but inhibited BSO cataract development. By contrast, 25 microM H-89, selective inhibitor of PKA, caused large annular cortical cataracts and 100-fold elevation of Na+/K+ within 30 hr in day 10 lenses, in either the presence or absence of BSO. H-89 cataracts were also seen in day 12 and day 21 lenses. 32P-labeling of day 12 lenses pretreated with H-89 displayed more than 80% decrease in phosphorylation of alphaA crystallin, a known substrate of PKA, in the insoluble protein fraction. 2D-gel electrophoresis of day 12 H-89 cataract lens fractions revealed limited degradation of alpha and beta crystallins, degradation of cytoskeletal proteins, and elevated lens Ca2+ (>4 nmol/mg wet wt.), suggesting Ca2+-activated proteolysis. Conclusions. High Na+/K+ cataracts are induced by H-89, selective inhibitor of PKA, but not by H-7, an inhibitor of both PKA and PKC that impeded BSO-induced Na+/K+ elevation and cataract. These results suggest contrasting effects of PKA and PKC on lens cation transport and cortical cataract development.     

Outcome of corneal transplant rejection: a 10-year study

PURPOSE: To study the incidence of graft rejection and the predictive factors for its reversibility. METHODS: It is a retrospective study of 1927 consecutive penetrating keratoplasties performed between January 1990 and January 2000 with more than 6 months follow up. A total of 224 rejection episodes were noted in 183 patients. Of these, 184 first rejection episodes were included in this study. RESULTS: The incidence of first rejection episode was 9.55%. Of patients 87% were symptomatic during the episode with vision loss being the commonest. The average time of onset of rejection was 15.25 +/- 14.4 months (median 11.7 months). In total, 53.3% of rejections occurred within 1 year after surgery. Of the patients who completed minimum 3 months follow up after the rejection episode, the rate of reversibility was 63.3%. Major predictive factors for a poor outcome after rejection were repeated grafting and associated anterior vitrectomy during surgery. The reversibility of the episode did not differ significantly with the modality of treatment used, but treatment with intravenous steroids within 7 days of onset of rejection may have a role in reducing the recurrences of rejection episodes, thus increasing the graft survival. CONCLUSION: Regrafts and associated anterior vitrectomy were significant risk factors for a poor outcome following rejection episode.     

The GM–CSF/IL-3/IL-5 cytokine receptor family: from ligand recognition to initiation of signaling

Granulocyte–macrophage colony-stimulating factor (GM–CSF), interleukin-3 (IL-3), and IL-5 are members of a discrete family of cytokines that regulates the growth, differentiation, migration and effector function activities of many hematopoietic cells and immunocytes. These cytokines are involved in normal responses to infectious agents, bridging innate and adaptive immunity. However, in certain cases, the overexpression of these cytokines or their receptors can lead to excessive or aberrant initiation of signaling resulting in pathological conditions, with chronic inflammatory diseases and myeloid leukemias the most notable examples. Recent crystal structures of the GM–CSF receptor ternary complex and the IL-5 binary complex have revealed new paradigms of cytokine receptor activation. Together with a wealth of associated structure–function studies, they have significantly enhanced our understanding of how these receptors recognize cytokines and initiate signals across cell membranes. Importantly, these structures provide opportunities for structure-based approaches for the discovery of novel and disease-specific therapeutics. In addition, recent biochemical evidence has suggested that the GM–CSF/IL-3/IL-5 receptor family is capable of interacting productively with other membrane proteins at the cell surface. Such interactions may afford additional or unique biological activities and might be harnessed for selective modulation of the function of these receptors in disease.     

JunD accentuates arecoline-induced disruption of tight junctions and promotes epithelial-to-mesenchymal transition by association with NEAT1 lncRNA

Head and neck cancers are highly prevalent in south-east Asia, primarily due to betel nut chewing. Arecoline, the primary alkaloid is highly carcinogenic; however its role in promoting tumorigenesis by disrupting junctional complexes and increasing risk of metastasis is not well delineated. Subsequently, the effects of low and high concentrations of arecoline on the stability of tight junctions and EMT induction were studied. A microarray analysis confirmed involvement of a MAPK component, JunD, in regulating tight junction-associated genes, specifically ZO-1. Results established that although arecoline-induced phosphorylation of JunD downregulated expression of ZO-1, JunD itself was modulated by the lncRNA-NEAT1 in presence of arecoline. Increased NEAT1 in tissues of HNSCC patients significantly correlated with poor disease prognosis. Here we show that NEAT1-JunD complex interacted with ZO-1 promoter in the nuclear compartment, downregulated expression of ZO-1 and destabilized tight junction assembly. Consequently, silencing NEAT1 in arecoline-exposed cells not only downregulated the expression of JunD and stabilized expression of ZO-1, but also reduced expression of the EMT markers, Slug and Snail, indicating its direct regulatory role in arecoline-mediated TJ disruption and disease progression.     

Dynamical network of residue–residue contacts reveals coupled allosteric effects in recognition,catalysis, and mutation

Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue–residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general.As a biomolecule samples various conformations governed by its free energy landscape, it undergoes a wide spectrum of motions that range over a broad timescale and length scale (1). Except for certain cases in which large-scale displacements are observed upon ligand binding (2), these motions, in general, account for modest fluctuations around an average native structure (3). Despite extensive studies, detailed understanding of how conformational dynamics lead to or facilitate function remains formidable (4–6). It has been frequently observed that enzymes in their free unliganded state sample 3D conformations that consist of those visited in the presence of the ligand (7). Differences in intrinsic conformational dynamics in the wild type and the mutant maltose-binding proteins have been shown to be related to association and dissociation of ligand and thereby to affect dissociation constants (8). Enzyme dynamics in Cyclophilin A (CypA), a peptidyl prolyl cis–trans isomerase, have been demonstrated to occur on the same millisecond timescale as the catalytic turnover (9). Mutants of dihydrofolate reductase that lead to suppressed conformational dynamics in the active site have exhibited concomitant loss in enzymatic activity (10).These exemplary studies are suggestive of a key role for conformational dynamics in recognition and catalysis. This notion has been controversial (10–13) due to limited or, in some cases, a complete lack of microscopic analysis of experimental observations. The suppressed catalytic activity in mutants may be a result of an increase in activation energy and not decreased dynamics (14, 15). Although enzyme dynamics may not be responsible for catalytic speed up relative to the uncatalyzed reaction, CypA dynamics has been shown to be coupled to catalytic function (16). Allosteric regulation, i.e., modification of binding or catalysis at the active site due to binding of a ligand at a distal nonoverlapping site, is widespread in biochemical signaling (17, 18). It is natural that allosteric regulation depends on modulation of protein motions, because substrate binding and catalysis are linked to conformational dynamics (19, 20). Unlike static X-ray structures, solution NMR relaxation dispersion techniques have been instrumental in providing high-resolution conformational exchange information using site-specific isotope labeling (21). In NMR studies of CypA, a dynamic continuum has been identified such that the relaxation profiles cannot be globally fit to one or two exchange phenomena and are instead indicative of more localized motions (22). Exchange rates coalesce somewhat during turnover, perhaps suggesting an increase in coordination throughout the protein, but appear to still consist of localized motions that are not fully coherent (23). However, dynamical signals during catalytic turnover could be affected by substrate binding and unbinding, especially if the substrate binding affinity is low, thereby leading to ambiguity in the interpretation of NMR analysis (23).Complementary to experiments, long-timescale molecular dynamics (MD) simulations can be greatly instrumental in providing a microscopic picture of biomolecular dynamics and establishing its exact linkage to function. However, the challenge at hand is to elucidate the key dynamical features and correlations between different parts of the protein from a vast amount of multidimensional time-dependent data from MD. Principal component analysis (PCA) is often used to reduce the dimensionality of conformational space and map the differences in conformational ensembles. PCA, which is usually performed on Cartesian coordinates, may sometimes mask certain important long-range dynamical relations and complex features of biomolecular conformational dynamics. Comparing residue–residue contacts in various isoforms from difference contact maps built from simulation trajectories has aided in determining certain similar structural properties and some unique to a particular isoform (24). Another analysis that involves monitoring the time evolution of residue–residue contact formation and breaking has been proven useful in identifying certain characteristic events during conformational transitions (25). Correlated motions between different biomolecular segments have been identified using cross-correlation analysis and building dynamical networks (25–29). However, interpretation of the results of such analysis in case of CypA, which exhibit subtle changes upon substrate binding and catalysis, has been ambiguous.Here, we characterize the conformational dynamics of CypA using very long atomistic standard MD simulations in explicit solvent, and the results are validated using NMR experiments. CypA is an archetypal and extensively studied enzyme belonging to the family of peptidyl prolyl isomerases (PPIases), speeding the cis–trans isomerization of peptidyl prolyl ω-bond in its protein substrates by more than 10⁵ times (30–34). In five independent microsecond-long simulations, we monitored the dynamics in wild-type CypA, V29L variant of CypA, CypA bound to a substrate analogue in the trans, transition state, and cis configurations. When we apply our method of analyzing the trajectories at a coarse-grained level, i.e., interresidue contact interactions, and use PCA in contact space, the specific differences in CypA dynamics upon association to its substrate, during the catalytic process and upon alteration of a single residue distant from the active site are revealed.