Bovine inositol monophosphatase: proteolysis and structural studies

AbstractBovine brain inositol monophosphatase is inactivated when trypsin catalyses the cleavage of a single peptide bond between Lys-36 and Ser-37. This proteolysis is closely followed by cleavage at two other sites in the protein between Lys-78 and Ser-79 and between Lys-156 and Ser-157 suggesting that all of these sites are exposed in the native conformation of the protein. All of these residues are predicted to lie at the ends of α helices. The most susceptible bond (Lys-36-Ser-37) is predicted to lie in a highly flexible region of the protein. Circular dichroism studies suggest that approximately 40% of the secondary structure of this protein is helical which is similar to that predicted by the algorithm of Gamier et al. [(1978) J. Mol. Biol. 120, 97-120]

Bovine inositol monophosphatase The identification of a histidine residue reactive to diethylpyrocarbonate

AbstractThe inositol monophosphatase from bovine brain is inactivated by the histidine-specific reagent diethylpyrocarbonate. Using 4 mM reagent at pH 6.5, the reaction results in the modification of 3 equivalents of histidine per polypeptide chain. The loss of activity occurs at the same rate as the slowest reacting of these residues. Site directed mutagenesis studies have been used to generate a mutated enzyme species bearing a His-217→Gln replacement and have shown that it is the modification of histidine 217 which results in the inactivation of the enzyme

Structural and functional studies on bovine and human brain inositol monophosphatase

SIGLEAvailable from British Library Document Supply Centre- DSC:DX180405 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Myc induces the nucleolin and BN51 genes: possible implications in ribosome biogenesis.

The c-Myc oncoprotein and its dimerization partner Max bind the DNA core consensus sequence CACGTG (E-box) and activate gene transcription. However, the low levels of induction have hindered the identification of novel Myc target genes by differential screening techniques. Here, we describe a computer-based pre-selection of candidate Myc/Max target genes, based on two restrictive criteria: an extended E-box consensus sequence for Myc/Max binding and the occurrence of this sequence within a potential genomic CpG island. Candidate genes selected by these criteria were evaluated experimentally for their response to Myc. Two Myc target genes are characterized here in detail. These encode nucleolin, an abundant nucleolar protein, and BN51, a co-factor of RNA polymerase III. Myc activates transcription of both genes via E-boxes located in their first introns, as seen for several well-characterized Myc targets. For both genes, mutation of the E-boxes abolishes transcriptional activation by Myc as well as repression by Mad1. In addition, the BN51 promoter is selectively activated by Myc and not by USF, another E-box-binding factor. Both nucleolin and BN51 are implicated in the maturation of ribosomal RNAs, albeit in different ways. We propose that Myc, via regulation of these and probably many other transcriptional targets, may be an important regulator of ribosome biogenesis

Co- and posttranslational modification of the alpha(1B)-adrenergic receptor: effects on receptor expression and function.

We have characterized the maturation, co- and posttranslational modifications, and functional properties of the alpha(1B)-adrenergic receptor (AR) expressed in different mammalian cells transfected using conventional approaches or the Semliki Forest virus system. We found that the alpha(1B)-AR undergoes N-linked glycosylation as demonstrated by its sensitivity to endoglycosidases and by the effect of tunicamycin on receptor maturation. Pulse-chase labeling experiments in BHK-21 cells demonstrate that the alpha(1B)-AR is synthesized as a 70 kDa core glycosylated precursor that is converted to the 90 kDa mature form of the receptor with a half-time of approximately 2 h. N-Linked glycosylation of the alpha(1B)-AR occurs at four asparagines on the N-terminus of the receptor. Mutations of the N-linked glycosylation sites did not have a significant effect on receptor function or expression. Surprisingly, receptor mutants lacking N-linked glycosylation migrated as heterogeneous bands in SDS-PAGE. Our findings demonstrate that N-linked glycosylation and phosphorylation, but not palmitoylation or O-linked glycosylation, contribute to the structural heterogeneity of the alpha(1B)-AR as it is observed in SDS-PAGE. The modifications found are similar in the different mammalian expression systems explored. Our findings indicate that the Semliki Forest virus system can provide large amounts of functional and fully glycosylated alpha(1B)-AR protein suitable for biochemical and structural studies. The results of this study contribute to elucidate the basic steps involved in the processing of G protein-coupled receptors as well as to optimize strategies for their overexpression

Mutagenesis and modelling of the alpha(1b)-adrenergic receptor highlight the role of the helix 3/helix 6 interface in receptor activation.

Computer simulations on a new model of the alpha1b-adrenergic receptor based on the crystal structure of rhodopsin have been combined with experimental mutagenesis to investigate the role of residues in the cytosolic half of helix 6 in receptor activation. Our results support the hypothesis that a salt bridge between the highly conserved arginine (R143(3.50)) of the E/DRY motif of helix 3 and a conserved glutamate (E289(6.30)) on helix 6 constrains the alpha1b-AR in the inactive state. In fact, mutations of E289(6.30) that weakened the R143(3.50)-E289(6.30) interaction constitutively activated the receptor. The functional effect of mutating other amino acids on helix 6 (F286(6.27), A292(6.33), L296(6.37), V299(6.40,) V300(6.41), and F303(6.44)) correlates with the extent of their interaction with helix 3 and in particular with R143(3.50) of the E/DRY sequence

The concept of spiritual care in mental health nursing

NoIn this paper we aim to clarify the issue of spiritual care in the context of mental health nursing. Background. The concept of spirituality in nursing has received a great deal of attention in recent years. However, despite many articles addressed to the issue, spiritual care remains poorly understood amongst nursing professionals and, as a result, spiritual needs are often neglected within the context of health care. Methods. A series of focus groups was conducted to obtain the views of service users, carers and mental health nursing professionals about the concept of spirituality and the provision of spiritual care in mental health nursing. Results. According to the views expressed in our focus groups, spiritual care relates to the acknowledgement of a person¿s sense of meaning and purpose to life which may, or may not, be expressed through formal religious beliefs and practices. The concept of spiritual care was also associated with the quality of interpersonal care in terms of the expression of love and compassion towards patients. Concerns were expressed that the ethos of mental health nursing and the atmosphere of care provision were becoming less personal, with increasing emphasis on the `mechanics of nursing¿. Conclusions. The perceived failure of service providers to attend adequately to this component of care may be symptomatic of a medical culture in which the more readily observable and measurable elements in care practice have assumed a prominence over the more subjective, deeply personal components. In order for staff to acknowledge these issues it is argued that a more holistic approach to care should be adopted, which would entail multidisciplinary education in spiritual care