Chloroplast culture VIII a new effect of kinetin in enhancing the synthesis and accumulation of protochlorophyllide invitro

By pretreating etiolated cucumber cotyledons with kinetin in the dark, it was observed that the plastids isolated from such tissues were 400% more active in the conversion of δ-aminolevulinic acid into protochlorophyllide, than plastids prepared from water-treated controls. The experimental evidence is consistent with the hypothesis that (a) the kinetin dark-pretreatment of the etiolated tissue, uncouples the joint biosynthesis of prothylakoids and protochlorophyll and results in the accumulation of excess prothylakoid membranes poorly supplied with protochlorophyllide (b) upon isolation of the plastids and incubation with δ-aminolevulinic acid, the latter is very rapidly converted into membrane-bound protochlorophyllide.     

The effect of post-translational modifications on the hemorrhagic activity of snake venom metalloproteinases

Metalloproteinases (MPs) are Zn(+)-dependent endoproteolytic enzymes, abundant in crotalid and viperid snake venoms. Most snake venom metalloproteinases (svMPs) are active on extracellular matrix components and this effect is thought to result in bleeding as a consequence of the basement membrane disruption in capillaries. Jararhagin and ACLH are hemorrhagic svMPs from Bothrops jararaca and Agkistrodon contortrix laticinctus venom, respectively. Both enzymes demonstrate proteolytic activity on fibrinogen and fibronectin and jararhagin inhibits collagen-induced platelet aggregation in vitro. This work describes the expression, purification and successful refolding of the recombinant ACLH zymogen (rPRO-ACLH) as well as the catalytic domain of jararhagin (rCDJARA). The heterologous proteins were produced in E. coli, an in vivo expression system that does not make post-translational modifications. The recombinant refolded proteins did not show any hemorrhagic activity in mice skin, as well as the native deglycosylated jararhagin and ACLH. However, they preserved their proteolytic activity on fibrinogen and fibronectin. It seems that the hemorrhagic properties of these hemorrhagins are dependent on post-translational modifications, whereas their proteolytic activity is not dependent on such modifications.     

Genetic Determinants of the Pre‐ and Post‐Azathioprine Therapy Thiopurine Methyltransferase Activity Phenotype

Thiopurine drug therapy has been reported to lead to a variable increase in red cell TPMT activity that may alter effective dose and therapeutic outcome. The aim of this study was to correlate Variable Number Tandem Repeat (VNTR) in the promoter region of the TPMT gene with induction of red cell TPMT activity in patients treated with azathioprine (AZA). In 58 patients, TPMT activity measured at 3 months was not significantly induced on average above pre‐therapy levels. Individual patients showed variation in TPMT activity pre‐ and post‐AZA therapy, however changes in TPMT activity were not predicted by VNTR configuration. In conclusion, TPMT promoter VNTRs are unlikely to play a significant role in changes in TPMT activity in response to AZA therapy.     

Phylogenetic Analysis of Different Breeds of Domestic Chickens in Selected Area of Peninsular Malaysia Inferred from Partial Cytochrome b Gene Information and RAPD Markers

The present investigation was carried out in an attempt to study the phylogenetic analysis of different breeds of domestic chickens in Peninsular Malaysia inferred from partial cytochrome b gene information and random amplified polymorphic DNA (RAPD) markers. Phylogenetic analysis using both neighbor-joining (NJ) and maximum parsimony (MP) methods produced three clusters that encompassed Type-I village chickens, the red jungle fowl subspecies and the Japanese Chunky broilers. The phylogenetic analysis also revealed that majority of the Malaysian commercial chickens were randomly assembled with the Type-II village chickens. In RAPD assay, phylogenetic analysis using neighbor-joining produced six clusters that were completely distinguished based on the locality of chickens. High levels of genetic variations were observed among the village chickens, the commercial broilers, and between the commercial broilers and layer chickens. In this study, it was found that Type-I village chickens could be distinguished from the commercial chickens and Type-II village chickens at the position of the 27th nucleotide of the 351 bp cytochrome b gene. This study also revealed that RAPD markers were unable to differentiate the type of chickens, but it showed the effectiveness of RAPD in evaluating the genetic variation and the genetic relationships between chicken lines and populations.     

Soft-sensing method based on FDLS-SVM in marine alkaline protease fermentation process

Abstract

To overcome the problem that soft-sensing model cannot be updated with the bioprocess changes, this article proposed a soft-sensing modeling method which combined fuzzy c-means clustering (FCM) algorithm with least squares support vector machine theory (LS-SVM). FCM is used for separating a whole training data set into several clusters with different centers, each subset is trained by LS-SVM and sub-models are developed to fit different hierarchical property of the process. The new sample data that bring new operation information is introduced in the model, and the fuzzy membership function of the sample to each clustering is first calculated by the FCM algorithm. Then, a corresponding LS-SVM sub-model of the clustering with the largest fuzzy membership function is used for performing dynamic learning so that the model can update online. The proposed method is applied to predict the key biological parameters in the marine alkaline protease MP process. The simulation result indicates that the soft-sensing modeling method increases the model’s adaptive abilities in various operation conditions and can improve its generalization ability.     

Oxidative stress,DNA damage,and the telomeric complex as therapeutic targets in acute neurodegeneration

Oxidative stress has been identified as an important contributor to neurodegeneration associated with acute CNS injuries and diseases such as spinal cord injury (SCI), traumatic brain injury (TBI), and ischemic stroke. In this review, we briefly detail the damaging effects of oxidative stress (lipid peroxidation, protein oxidation, etc.) with a particular emphasis on DNA damage. Evidence for DNA damage in acute CNS injuries is presented along with its downstream effects on neuronal viability. In particular, unchecked oxidative DNA damage initiates a series of signaling events (e.g. activation of p53 and PARP-1, cell cycle re-activation) which have been shown to promote neuronal loss following CNS injury. These findings suggest that preventing DNA damage might be an effective way to promote neuronal survival and enhance neurological recovery in these conditions. Finally, we identify the telomere and telomere-associated proteins (e.g. telomerase) as novel therapeutic targets in the treatment of neurodegeneration due to their ability to modulate the neuronal response to both oxidative stress and DNA damage.     

Accurate ab initio calculation of the Ar–CF4 intermolecular potential energy surface

Ar–CF₄ intermolecular interaction potential is studied by ab initio calculations at the MP2 and CCSD(T) levels of theory containing the so-called bond functions ({3s3p2d1f} basis set was chosen) both with and without a correction for the basis-set superposition error. The calculations were performed with Dunning's correlation consistent basis sets (aug-cc-pVXZ, X = D, T, Q, 5) to extrapolate the Ar–CF₄ potential energy minimum and intermolecular distance to their complete basis set (CBS) limits. It is shown that the addition of bond functions results in a dramatic improvement in the convergence of the calculated interaction energies at the MP2/aug-cc-pVTZ level. The MP2/{3s3p2d1f}-aug-cc-pVTZ potential energy surface even approaches the CCSD(T)/aug-cc-pVQZ potential energy surface. The potential energy minima and the intermolecular distances are both significantly closer to the CBS limit when using the bond functions, and it implies that adding bond functions in the calculation has a great effect on the interaction energies. We also find that with bond functions included in the CCSD(T)/aug-cc-pVDZ model chemistry, the potential energy minima are extremely close to the CBS limit and are better than the CCSD(T)/aug-cc-pVQZ values. Several levels of theory described in the text were used to determine pairwise analytic potential energy surfaces for Ar+CF₄. The analytic potential energy surfaces are in very good agreement with the ab initio values.     

Ubiquitination and downregulation of ErbB2 and estrogen receptor-alpha by kinase inhibitor MP-412 in human breast cancer cells

ErbB2 has been proven to be an important target for breast cancer therapy. MP-412 is a dual ErbB2 and epidermal growth factor receptor tyrosine kinase inhibitor belonging to an irreversible-type anilinoquinazoline derivative. We demonstrate herein that along with the kinase inhibition, MP-412 has the ability to induce ubiquitination, internalization, and degradation of ErbB2 in several human breast cancer cell lines at concentrations relatively higher than those required for kinase inhibition. Another irreversible inhibitor, CI-1033, showed similar activity, while the reversible compounds were ineffective, suggesting a crucial role of covalent bonding functionality in these effects. In MCF7 cells, MP-412 depleted not only ErbB2 but also estrogen receptor (ER)-α, and to some extent, affected Raf-1, while MP-412 activated Hsp70 expression. Moreover, we observed that MP-412 increased immunocomplexing of Hsp70 with ErbB2 and ER-α, with simultaneous induction of ubiquitination of these client proteins. Furthermore, in combination with proteasome inhibitor, MP-412 resulted in the noticeable accumulation of ErbB2 and ER-α in the detergent insoluble fraction of cell lysates. These results suggest that MP-412 acts as an inhibitor of Hsp90 function, whereas MP-412 did not bind directly to ATP-binding site of Hsp90, unlike geldanamycin. We also found that new protein synthesis was involved in the activity of MP-412 on Hsp90 modulation. Since downregulation of ErbB2 and ER-α by accelerating the ubiquitin-proteolysis system will become an attractive approach for breast cancer therapy, we expect MP-412 to be a lead compound for the drug design and the development of such agents.     

Discordances between phylogenetic and morphological patterns in alpine leaf beetles attest to an intricate biogeographic history of lineages in postglacial Europe

Pleistocene glacial and interglacial periods have moulded the evolutionary history of European cold-adapted organisms. The role of the different mountain massifs has, however, not been accurately investigated in the case of high-altitude insect species. Here, we focus on three closely related species of non-flying leaf beetles of the genus Oreina (Coleoptera, Chrysomelidae), which are often found in sympatry within the mountain ranges of Europe. After showing that the species concept as currently applied does not match barcoding results, we show, based on more than 700 sequences from one nuclear and three mitochondrial genes, the role of biogeography in shaping the phylogenetic hypothesis. Dating the phylogeny using an insect molecular clock, we show that the earliest lineages diverged more than 1 Mya and that the main shift in diversification rate occurred between 0.36 and 0.18 Mya. By using a probabilistic approach on the parsimony-based dispersal/vicariance framework (MP-DIVA) as well as a direct likelihood method of state change optimization, we show that the Alps acted as a cross-roads with multiple events of dispersal to and reinvasion from neighbouring mountains. However, the relative importance of vicariance vs. dispersal events on the process of rapid diversification remains difficult to evaluate because of a bias towards overestimation of vicariance in the DIVA algorithm. Parallels are drawn with recent studies of cold-adapted species, although our study reveals novel patterns in diversity and genetic links between European mountains, and highlights the importance of neglected regions, such as the Jura and the Balkanic range.     

Evidence for the location of the allosteric activation switch in the multisubunit phosphorylase kinase complex from mass spectrometric identification of chemically crosslinked peptides

Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, regulates glycogenolysis. Its activity, catalyzed by the gamma subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory alpha, beta and delta subunits. Activation by phosphorylation is predominantly mediated by the regulatory beta subunit, which undergoes a conformational change that is structurally linked with the gamma subunit and that is characterized by the ability of a short chemical crosslinker to form beta-beta dimers. To determine potential regions of interaction of the beta and gamma subunits, we have used chemical crosslinking and two-hybrid screening. The beta and gamma subunits were crosslinked to each other in phosphorylated PhK, and crosslinked peptides from digests were identified by Fourier transform mass spectrometry, beginning with a search engine developed "in house" that generates a hypothetical list of crosslinked peptides. A conjugate between beta and gamma that was verified by MS/MS corresponded to crosslinking between K303 in the C-terminal regulatory domain of gamma (gammaCRD) and R18 in the N-terminal regulatory region of beta (beta1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of beta inhibited the crosslinking between beta and gamma, and was itself crosslinked to K303 of gamma. In two-hybrid screening, the beta1-31 region controlled beta subunit self-interactions, in that they were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the beta and gamma subunits. The sum of our results considered together with previous findings implicates the gammaCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme's regulatory subunits and is proximal to the active site cleft.