Molecular details of Itk activation by prolyl isomerization and phospholigand binding: the NMR structure of the Itk SH2 domain bound to a phosphopeptide

The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) is a critical component of the regulatory apparatus controlling the activity of this immunologically important enzyme. To gain insight into the structural features associated with the activated form of Itk, we have solved the NMR structure of the SH2 domain bound to a phosphotyrosine-containing peptide (pY) and analyzed changes in trans-hydrogen bond scalar couplings ((3h)J(NC')) that result from pY binding. Isomerization of a single prolyl imide bond in this domain is responsible for simultaneous existence of two distinct SH2 conformers. Prolyl isomerization directs ligand recognition: the trans conformer preferentially binds pY. The structure of the SH2/pY complex provides insight into the ligand specificity; the BG loop in the ligand-free trans SH2 conformer is pre-arranged for optimal contacts with the pY+3 residue of the ligand. Analysis of (3h)J(NC') couplings arising from hydrogen bonds has revealed propagation of structural changes from the pY binding pocket to the CD loop containing conformationally heterogeneous proline as well as to the alphaB helix, on the opposite site of the domain. These findings offer a structural framework for understanding the roles of prolyl isomerization and pY binding in Itk regulation.     

Cernunnos interacts with the XRCC4 x DNA-ligase IV complex and is homologous to the yeast nonhomologous end-joining factor Nej1

DNA double strand breaks are considered as the most harmful DNA lesions and are repaired by either homologous recombination or nonhomologous end joining (NHEJ). A new NHEJ factor, Cernunnos, has been identified, the defect of which leads to a severe immunodeficiency condition associated with microcephaly and other developmental defects in humans. This presentation is reminiscent to that of DNA-ligase IV deficiency and suggests a possible interplay between Cernunnos and the XRCC4 x DNA-ligase IV complex. We show here that Cernunnos physically interacts with the XRCC4 x DNA-ligase IV complex. Moreover, a combination of sensitive methods of sequence analysis revealed that Cernunnos can be associated with the XRCC4 family of proteins and that it corresponds to the genuine homolog of the yeast Nej1 protein. Altogether these results shed new lights on the last step, the DNA religation, of the NHEJ pathway.     

SMN-mediated assembly of RNPs: a complex story

Although many RNA-protein complexes or ribonucleoproteins (RNPs) assemble spontaneously in vitro, little is known about how they form in the environment of a living cell. Insight into RNP assembly has come unexpectedly from functional analyses of the survival motor neuron (SMN) protein, a gene product that is affected in the neuromuscular disease spinal muscular atrophy. These studies show that the assembly of spliceosomal U-rich small nuclear RNPs in vivo depends on the activity of two large protein complexes, one of which contains the SMN protein. These complexes might also facilitate the assembly of other cellular RNPs.     

A technique for joint center analysis using a stored program calculator

For a long time in the study of joint kinematics, the instant center of rotation in plane motion was obtained through graphic drawings. Since then the study of joint kinematics has become three-dimensional involving the use of computers. For this paper a stored-program calculator has been used as it is a precise instrument and several films can be used even if their positions are very close to one another.

A movement is never perfectly plane, it was important to define a coefficient (in percentage) to qualify the more or less plane character of a movement.

We believe that an analytical location is a better way than using graphic drawings of I.C.R.:

1. (1) to smooth the raw coordinates;

2. (2) to calculate the plane motion coefficient in order to eliminate an X-Ray picture of a whole series of pictures for lack of plane character;

3. (3) to define in the results an error rectangle whose dimensions are linked to errors in the observation and then to pick out among the points of a body those with the smallest risk for error.

To probe this method the two radio-ulnaris joints have been studied. At present studies are being carried on to compare the I.C.R.'s behaviour of the lumbar spine during a motion of lateral inflexion both in the case of normal people and people with scoliosis.     

Apoplastic and cytosolic expression of full‐size antibodies and antibody fragments in Nicotiana tabacum

We compared the expression of a functional recombinant TMVspecific fullsize antibody (rAb29) in both the apoplast and cytosol of tobacco plants and a single chain antibody fragment (scFv29), derived from rAb29, was expressed in the cytosol. Cloned heavy and light chain cDNAs of fullsize rAb29, which binds to TMV coat protein monomers, were integrated into the plant expression vector pSS. The fullsize rAb29 was expressed in the cytosol and targeted to the apoplast by including the original murine antibody leader sequences. Levels of functional fullsize rAb29 expression were high in the apoplast (up to 8.5g per gram leaf tissue), whereas cytosolic expression was low or at the ELISA detection limit. Sequences of the variable domains of rAb29 light and heavy chain were used to generate the single chain antibody scFv29, which was expressed in the periplasmic space of E.coli and showed the same binding specificity as fullsize rAb29. In addition, scFv29 was functionally expressed in the cytosol of tobacco plants and plant derived scFv29 maintained same binding specificity to TMVcoat protein monomers as rAb29.     

Amplification of CRAC current by STIM1 and CRACM1 (Orai1)

Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (I(CRAC)). However, individual overexpression of either protein fails to significantly amplify I(CRAC). Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates I(CRAC), suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.     

Biosynthesis of riboflavin in archaea. 6,7-dimethyl-8-ribityllumazine synthase of Methanococcus jannaschii.

Heterologous expression of the putative open reading frame MJ0303 of Methanococcus jannaschii provided a recombinant protein catalysing the formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Steady state kinetic analysis at 37 degrees C and pH 7.0 indicated a catalytic rate of 11 nmol.mg-1.min-1; Km values for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxybutanone 4-phosphate were 12.5 and 52 micro m, respectively. The enzyme sediments at an apparent velocity of about 12 S. Sedimentation equilibrium analysis indicated a molecular mass around 1 MDa but was hampered by nonideal solute behaviour. Negative-stained electron micrographs showed predominantly spherical particles with a diameter of about 150 A. The data suggest that the enzyme from M. jannaschii can form capsids with icosahedral 532 symmetry consisting of 60 subunits.     

A novel Fomitiporia species associated with esca on grapevine in South Africa

Esca disease of grapevine is a complex trunk disease consisting of several symptoms, one of which, white rot, has been found to be caused by various basidiomycetes within the order Hymenochaetales. During recent surveys of esca-related pathogens in South African vineyards, several unidentified basidiomycetes were isolated from white rot occurring in diseased vines. A new Fomitiporia species, F. capensis, is described based on fruit body morphology and combined internal transcribed spacer (ITS) and large-subunit (LSU) phylogeny, where it forms a clearly delineated and well-supported clade. Morphologically, F. capensis is similar to F. punctata in that both species essentially lack setae. Fomitiporia capensis, F. punctata and F. aethiopica produce similarly sized basidiospores but differ in terms of host range, pore size and, possibly, fruit body shape. Phylogenetically, F. capensis appears to be related to F. tenuis, though morphologically, the species differ significantly in that F. tenuis has smaller pores and smaller basidiospores. Fomitiporia capensis was found to occur widely as vegetative mycelium throughout the Western Cape Province, though fruit bodies were scarce in comparison. A vineyard with fruit bodies was also found in Limpopo in the north east of the country. Fruit bodies were found growing on the underside of the cordon of living vines displaying external symptoms typically associated with esca, or general decline and dieback symptoms together with internal white rot.     

Impact of ABCB1 and CYP2B6 Genetic Polymorphisms on Methadone Metabolism,Dose and Treatment Response in Patients with Opioid Addiction: A Systematic Review and Meta-Analysis

Background

Genetic variability may influence methadone metabolism, dose requirements, and risk of relapse.

Objectives

To determine whether the CYP2B6*6 or ABCB1 (rs1045642) polymorphisms are associated with variation in methadone response (plasma concentration, dose, or response to treatment).

Methods

Two independent reviewers searched Medline, EMBASE, CINAHL, PsycINFO, and Web of Science databases. We included studies that reported methadone plasma concentration, methadone response, or methadone dose in relation to the CYP2B6*6 or ABCB1 polymorphisms.

Results

We screened 182 articles and extracted 7 articles for inclusion in the meta-analysis. Considerable agreement was observed between the two independent raters on the title (kappa, 0.82), abstract (kappa, 0.43), and full text screening (kappa, 0.43). Trough (R) methadone plasma concentration was significantly higher in CYP2B6*6 homozygous carriers when compared to non-carriers (standardized mean difference [SMD] = 0.53, 95% confidence interval [CI], 0.05–1.00, p = 0.03) with minimal heterogeneity (I² = 0%). Similarly, trough (S) methadone plasma concentration was higher in homozygous carriers of the *6 haplotype when compared to non-carriers, (SMD = 1.44, 95% CI 0.27–2.61, p = 0.02) however significant heterogeneity was observed (I² = 69%). Carriers of the CYP2B6*6 haplotype were not found to be significantly different from non-carriers with respect to dose or response to treatment. We found no significant association between the ABCB1 polymorphism and the trough (R), (S) plasma concentrations, methadone dose, or methadone response.

Conclusion

Although the number of studies included and sample size were modest, this is the first meta analysis to show participants homozygous for the CYP2B6*6 genotype have higher trough (R) and (S) methadone plasma concentrations, suggesting that methadone metabolism is significantly slower in *6 homozygous carriers.