Molecular mechanisms of protein kinase-mediated desensitization and internalization of the $\beta$-adrenergic receptor

The $\beta$-adrenergic receptor ($\beta$AR), which couples to G$\sb{\rm s}$ and activates adenylylcyclase, has been a prototype for studying the activation and desensitization of G-protein-coupled receptors. The main objective of the present study is to elucidate the molecular mechanisms of protein kinase-mediated desensitization and internalization of the $\beta$AR.^ Activation of cAPK or PKC causes a rapid desensitization of $\beta$AR stimulation of adenylylcyclase in L cells, which previous studies suggest involves the cAPK/PKC consensus phosphorylation site in the third intracellular loop of the $\beta$AR, RRSSK$\sp{263}$. To determine the role of the individual serines in the cAPK- and PKC-meditated desensitizations, wild type (WT) and mutant $\beta$ARs containing the substitutions, Ser$\sp{261} \to$ A, Ser$\sp{262} \to$ A, Ser$\sp{262} \to$ D, and Ser$\sp{261/262} \to$ A, were constructed and stably transfected into L cells. The cAPK-mediated desensitization was decreased 70-80% by the Ser$\sp{262} \to$ A, Ser$\sp{262} \to$ D, and the Ser$\sp{261/262} \to$ A mutations, but was not altered by the Ser$\sp{261} \to$ A substitution, demonstrating that Ser$\sp{262}$ was the primary site of the cAPK-induced desensitization. The PMA/PKC-induced desensitization was unaffected by either of the single serine to alanine substitutions, but was reduced 80% by the double serine to alanine substitution, suggesting that either serine was sufficient to confer the PKC-mediated desensitization. Coincident stimulation of cAPK and PKC caused an additive desensitization which was significantly reduced (80%) only by the double substitution mutation. Quantitative evaluation of the coupling efficiencies and the GTP-shift of the WT and mutant receptors demonstrated that only one of the mutants, Ser$\sp{262} \to$ A, was partially uncoupled. The Ser$\sp{262} \to$ D mutation did not significantly uncouple, demonstrating that introducing a negative charge did not appear to mimic the desensitized state of the receptor.^ To accomplish the in vivo phosphorylation of the $\beta$AR, we used two epitope-modified $\beta$ARs, hemagglutinin-tagged $\beta$AR (HA-$\beta$AR) and 6 histidine-tagged $\beta$AR (6His-$\beta$AR), for a high efficiency purification of the $\beta$AR. Neither HA-$\beta$AR nor 6His-$\beta$AR altered activation and desensitization of the $\beta$AR significantly as compared to unmodified wild type $\beta$AR. 61% recovery of ICYP-labeled $\beta$AR was obtained with Ni-NTA column chromatography.^ The truncation 354 mutant $\beta$AR(T354), lacking putative $\beta$ARK site(s), displayed a normal epinephrine stimulation of adenylylcyclase. Although 1.0 $\mu$M epinephrine induced 60% less desensitization in T354 as compared to wild type $\beta$AR, 1.0 $\mu$M epinephrine-mediated desensitization in T354 was 35% greater than PGE$\sb1$-mediated desensitization, which is essentially identical in both WT and T354. These results suggested that sequences downstream of residue 354 may play a role in homologous desensitization and that internalization may be attributed to the additional desensitization besides the cAMP mechanism in T354 $\beta$AR. (Abstract shortened by UMI.) ^

Remote Effects Modulating the Spin Equilibrium of the Resting State of Cytochrome P450cam –An Investigation Using Active Site Analogues

The crystal structure of the resting state of cytochrome P450cam (CYP101), a heme thiolate protein, shows a cluster of six water molecules in the substrate binding pocket, one of which is coordinating to iron(III) as sixth ligand. The resting state is low-spin and changes to high-spin when substrate camphor binds and H2O is removed. In contrast to the protein, previously synthesised enzyme models such as H2O[BOND]FeIII(porph)(ArS−) were shown to be purely high-spin. Iron(S−)porphyrins with different distal sites mimicking proposed remote effects have been prepared and studied by cw-EPR. The results indicate that the low-spin of the resting state of P450cam is due to the fact that the water molecule coordinating to iron has an OH−-like character because of hydrogen bonding and polarisation of the water cluster, respectively.

"Quasi ombra e figura de la verità". Poesia e pensiero in Torquato Tasso

"Pochi autori come il Tasso hanno suscitato interpretazioni tanto differenziate e, in molti casi, discordanti. Posto di volta in volta a chiusura di un'epoca, quella manieristica, o come anticipatore della successiva temperie barocca, egli pare aver scontato il prezzo di un'esemplarità sin troppo manifesta rispetto al proprio Zeitgeist, una stagione che vede, nella vulgata storiografica, la regola prevalere sull'afflato creativo. 'Era già critico prima di esser poeta', sentenziò il De Sanctis, che arrivava a circoscriverne pure l'esperienza biografica nel perimetro di un implacabile autodafé, definendolo 'più crudele inquisitore di sé che il tribunale dell'Inquisizione'. A ciò poi si aggiungeva l'estrinsecità della corte quale destinataria e quasi principio informativo del mondo tassiano, che sembrava legittimare, da Croce in avanti, tutta una serie di giudizi limitativi sulla sua poesia, orientati ad accentuarne la natura encomiastica, aneddotica, addirittura frivola. La critica più recente ha compiuto un'imponente e fondamentale opera di scavo all'interno della cultura tassiana, delle sue letture, della sua poetica, ma il tentativo di rettificare il profilo complessivo dell'autore mostra esiti ancora incerti. In risposta alla vecchia immagine di un Tasso sconfitto dalla cultura controriformistica e alla fine assoggettato alle sue leggi, se ne sono di recente aggiunte altre..." (Dall'introduzione)

A methodology for appraising and selecting strategies to mitigate desertification based on stakeholder participation and global best practices

The main aim of the methodology presented in this paper is to provide a framework for a participatory process for the appraisal and selection of options to mitigate desertification and land degradation. This methodology is being developed within the EU project DESIRE (www.desire-project.eu/) in collaboration with WOCAT (www.wocat.org). It is used to select promising conservation strategies for test-implementation in each of the 16 degradation and desertification hotspot sites in the Mediterranean and around the world. The methodology consists of three main parts: In a first step, prevention and mitigation strategies already applied at the respective DESIRE study site are identified and listed during a workshop with representatives of different stakeholders groups (land users, policy makers, researchers). The participatory and process-oriented approach initiates a mutual learning process among the different stakeholders by sharing knowledge and jointly reflecting on current problems and solutions related to land degradation and desertification. In the second step these identified, locally applied solutions (technologies and approaches) are assessed with the help of the WOCAT methodology. Comprehensive questionnaires and a database system have been developed to document and evaluate all relevant aspects of technical measures as well as implementation approaches by teams of researchers and specialists, together with land users. This research process ensures systematic assessing and piecing together of local information, together with specific details about the environmental and socio-economic setting. The third part consists of another stakeholder workshop where promising strategies for sustainable land management in the given context are selected, based on the best practices database of WOCAT, including the evaluated locally applied strategies at the DESIRE sites. These promising strategies will be assessed with the help of a selection and decision support tool and adapted for test-implementation at the study site.

The role of LMX1B and PITX2 in anterior segment development and adult ocular function

Several congenital syndromes associated with anterior segment (AS) anomalies can lead to impaired vision and glaucoma, such as nail-patella syndrome (NPS), caused by mutations in the LIM homeodomain transcription factor LMX1B and Axenfeld-Rieger's syndrome (ARS), caused by mutations in the bicoid-related homeodomain transcription factor PITX2. Targeted mutations in lmx1b and pitx2 and RNA in situ analysis reveal that both genes are required for AS development and are co-expressed within the periocular mesenchyme, suggesting they participate in a shared genetic pathway. Lmx1b homozygous mutants display iris and corneal stroma hypoplasia, and defects in ciliary body formation. In contrast, pitx2 homozygous mutants exhibit a more severe phenotype: the AS chamber, corneal endothelium, and extraocular muscles (EOM) fail to develop. The absence of EOM in pitx2 mutants suggests pitx2 acts upstream of lmx1b, or that other lmx1b family members, such as lmx1a, can compensate for lmx1b function. Lmxla/lmx1b double homozygous mutants have a reduced capacity to generate EOM, implying that lmx1 gene products have a redundant function in EOM development and that lmx1 family members may act downstream of pitx2. However, analysis of pitx2 expression in the AS tissues of lmx1b mutants and reciprocal studies of lmx1b expression in pitx2 mutants indicate that these genes do not function in a simple linear pathway. Instead, lmx1b and pitx2 may regulate a shared set of downstream targets or both genes may work in parallel transcribing unique targets required for a common biological process. Ultrastructural analysis of lmx1b and pitx2 mutant corneas indicates that collagen fibrillogenesis is perturbed, revealing a common role for both genes in the deposition of extracellular matrix. Furthermore, lmx1b/pitx2 double heterozygotes develop corneal opacities not observed in single heterozygotes demonstrating that lmx1b and pitx2 genetically interact. Data suggests that defects in the basement membrane of the corneal endothelium underlie the opacities observed in double heterozygotes. Additionally, double heterozygotes develop anterior synechias that occlude the trabecular meshwork, potentially blocking aqueous humor drainage. These data suggest that lmx1b and pitx2 are responsible for ECM deposition in multiple cell types and imply that such defects may contribute to the glaucomas observed in NPS and ARS patients. ^