Экономические перспективы повышения уровня использования попутного нефтяного газа

A novel method employing filter arrays of a cDNA expression library for the identification of substrates for protein kinases was developed. With this technique, we identified a new member of the cyclin family, cyclin L2, as a substrate of the nuclear protein kinase DYRK1A. Cyclin L2 contains an N-terminal cyclin domain and a C-terminal arginine/serine-rich domain (RS domain), which is a hallmark of many proteins involved in pre-mRNA processing. The gene for cyclin L2 encodes the full-length cyclin L2, which is predominantly expressed in testis, as well as a truncated splicing variant (cyclin L2S) that lacks the RS domain and is ubiquitously expressed in human tissues. Full-length cyclin L2, but not cyclin L2S, was associated with the cyclin-dependent kinase PITSLRE. Cyclin L2 interacted with splicing factor 2 in vitro and was co-localized with the splicing factor SC35 in the nuclear speckle compartment. Photobleaching experiments showed that a fusion protein of green fluorescent protein and cyclin L2 in nuclear speckles rapidly exchanged with unbleached molecules in the nucleus, similar to other RS domain-containing proteins. In striking contrast, the closely related green fluorescent protein-cyclin L1 was immobile in the speckle compartment. DYRK1A interacted with cyclin L2 in pull-down assays, and overexpression of DYRK1A stimulated phosphorylation of cyclin L2 in COS-7 cells. These data characterize cyclin L2 as a highly mobile component of nuclear speckles and suggest that DYRK1A may regulate splicing by phosphorylation of cyclin L2

The recognition of collagen and triple-helical toolkit peptides by MMP-13: sequence specificity for binding and cleavage.

Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly(775)-Leu(776) in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cleavage site. MMP-13 was unable to bind to a linear peptide of the same sequence as II-44. We also discovered a second binding site near the N terminus of collagen II (starting at helix residue 127) in Toolkit peptide II-8. The pattern of binding of the free hemopexin domain of MMP-13 was similar to that of the full-length enzyme, but the free catalytic subunit bound none of our peptides. The susceptibility of Toolkit peptides to proteolysis in solution was independent of the very specific recognition of immobilized peptides by MMP-13; the enzyme proved able to cleave a range of dissolved collagen peptides.This work was supported by a British Heart Foundation programme grant, RG/009/003/27122, and peptide synthesis, by grants from Medical Research Council and Wellcome Trust.This is the author accepted manuscript. The final version can be found on the publisher's website at: http://www.jbc.org/content/early/2014/07/09/jbc.M114.58344

Mutational Analysis of Bacillus megaterium QM B1551 Cortex-Lytic Enzymes ▿ †

Molecular-genetic and muropeptide analysis techniques have been applied to examine the function in vivo of the Bacillus megaterium QM B1551 SleB and SleL proteins. In common with Bacillus subtilis and Bacillus anthracis, the presence of anhydromuropeptides in B. megaterium germination exudates, which is indicative of lytic transglycosylase activity, is associated with an intact sleB structural gene. B. megaterium sleB cwlJ double mutant strains complemented with engineered SleB variants in which the predicted N- or C-terminal domain has been deleted (SleB-ΔN or SleB-ΔC) efficiently initiate and hydrolyze the cortex, generating anhydromuropeptides in the process. Additionally, sleB cwlJ strains complemented with SleB-ΔN or SleB-ΔC, in which glutamate and aspartate residues have individually been changed to alanine, all retain the ability to hydrolyze the cortex to various degrees during germination, with concomitant release of anhydromuropeptides to the surrounding medium. These data indicate that while the presence of either the N- or C-terminal domain of B. megaterium SleB is sufficient for initiation of cortex hydrolysis and the generation of anhydromuropeptides, the perceived lytic transglycosylase activity may be derived from an enzyme(s), perhaps exclusively or in addition to SleB, which has yet to be identified. B. megaterium SleL appears to be associated with the epimerase-type activity observed previously in B. subtilis, differing from the glucosaminidase function that is apparent in B. cereus/B. anthracis

Characterization of cyclin L2, a novel cyclin with an arginine/serine-rich domain : phosphorylation by DYRK1A and colocalization with splicing factors

A novel method employing filter arrays of a cDNA expression library for the identification of substrates for protein kinases was developed. With this technique, we identified a new member of the cyclin family, cyclin L2, as a substrate of the nuclear protein kinase DYRK1A. Cyclin L2 contains an N-terminal cyclin domain and a C-terminal arginine/serine-rich domain (RS domain), which is a hallmark of many proteins involved in pre-mRNA processing. The gene for cyclin L2 encodes the full-length cyclin L2, which is predominantly expressed in testis, as well as a truncated splicing variant (cyclin L2S) that lacks the RS domain and is ubiquitously expressed in human tissues. Full-length cyclin L2, but not cyclin L2S, was associated with the cyclin-dependent kinase PITSLRE. Cyclin L2 interacted with splicing factor 2 in vitro and was co-localized with the splicing factor SC35 in the nuclear speckle compartment. Photobleaching experiments showed that a fusion protein of green fluorescent protein and cyclin L2 in nuclear speckles rapidly exchanged with unbleached molecules in the nucleus, similar to other RS domain-containing proteins. In striking contrast, the closely related green fluorescent protein-cyclin L1 was immobile in the speckle compartment. DYRK1A interacted with cyclin L2 in pull-down assays, and overexpression of DYRK1A stimulated phosphorylation of cyclin L2 in COS-7 cells. These data characterize cyclin L2 as a highly mobile component of nuclear speckles and suggest that DYRK1A may regulate splicing by phosphorylation of cyclin L2

Geologia, geoquímica e afinidades tectonomagmáticas dos granitoides de Bannach do Domínio Rio Maria, Província Carajás, Brasil

A área a norte de Bannach, Pará, é formada por trondhjemitos, leucogranodioritos, biotita granodioritos, tonalitos com anfibólio (± quartzo dioritos), biotita tonalitos (enclaves) e granitoides finos. O grupo de rochas com alta sílica (> 70% SiO2), representado pelos trondhjemitos, leucogranodioritos e granitoides finos, apresenta altos teores de Al2O3, CaO e Na2O em detrimento ao Fe2O3, MgO, Ni e Cr. Esse grupo exibe elevadas razões La/Y e padrão elementos terras raras (ETR) fortemente fracionado, caracterizado pela ausência de anomalias negativas de Eu. Os leucogranodioritos apresentam enriquecimento em Na2O, Ba e Sr. Os granitoides de baixa sílica, representados pelos biotita granodiorito, tonalito com anfibólio e enclaves tonalíticos apresentam alto conteúdo de Fe2O3, MgO, Ni e Y. Os biotita granodioritos apresentam altos teores de Ba-Sr-K e moderadas a altas razões La/Yb, enquanto os demais apresentam baixas razões La/Yb. A origem dos trondhjemitos é atribuída à fusão parcial de granada anfibolitos em ambiente de subducção e os leucogranodioritos têm seu controle composicional associado a diferentes graus de fusão de basaltos enriquecidos da base da crosta. O caráter geoquímico ambíguo dos biotita granodioritos (altos conteúdos de Rb e Ba, Sr, Y e razões La/Y e Sr/Y elevadas) os aproxima dos granitos tipo Closepet. Por sua vez, o caráter menos evoluído dos tonalitos com anfibólio, bem como as baixas razões La/Yb e Sr/Y, indicam que estas possuem afinidade mantélica e que foram formadas em baixas profundidades. Admite-se que a origem dessas rochas esteja relacionada ao manto metassomatizado por fluidos da slab em ambiente de subducção.