Dynamics of Newly Synthesized RNA and Protein Diffusion from Germinating Pollen of Cfivia nobilis Cultured In Vitro

Release of newly synthesized RNA (NSRNA) and protein (NSP) from.germinating pollen(GP) of Clivia nobilis was observed and proven by using autoradiograph, labeled with 3H-uridine and 3H-leucine, and treating with inhibitors (Actinimycin D and Cycloheximide). Its dynamics are as follows: 1 h after labeling before the germination of the pollens, a large amount of NSRNA and NSP were synthesized already. 2 hrs later, the NSP transported towards PT. 5 hrs later, it diffused into the growth medium. The NSRNA .concentrated.in vegetative nucleus and generative cells with in the first 90 min. 2 hrs later it diffused to the protoplasm of the pollens, 3 hrs later-concentrated on the tip of PT. 5 hrs laterout of GP. The biological significance of the phenomenon was discussed.     

人α－肿瘤坏死因子基因的高效表达

用人工合成的α－肿瘤坏死因子（ＴＮＦ－α）基因构建了五个不同的表达质粒,它们不同之处是ＳＤ序列与起始密码子ＡＴＧ间距离（Ｄ）各异。计算机模拟计算出翻译起始区域（ＴＩＲ）中二级结构的最小生成自由能,以（Ｄ）为６个核苷酸时最小（绝对值）。它的表达效率也最高,产物ＴＮＦ－α可达菌体总蛋白的６０％。密码子的选用对表达效率有很大的影响,故人工合成ＴＮＦ－α基因（选用大肠杆菌偏爱的密码子）的表达效率高于ｓｃ－     

Dynamics of granzyme B-induced apoptosis: mathematical modeling

Apoptosis is mediated by an intracellular biochemical system that mainly includes proteins (procaspases, caspases, inhibitors, Bcl-2 protein family as well as substances released from mitochondrial intermembrane space). The dynamics of caspase activation and target cleavage in apoptosis induced by granzyme B in a single K562 cell was studied using a mathematical model of the dynamics of granzyme B-induced apoptosis developed in this work. Also the first application of optimization approach to determination of unknown kinetic constants of biochemical apoptotic reactions was presented. The optimization approach involves solving of two problems: direct and inverse. Solving the direct optimization problem, we obtain the initial (baseline) concentrations of procaspases for known kinetic constants through conditional minimization of a cost function based on the principle of minimum protein consumption by the apoptosis system. The inverse optimization problem is aimed at determination of unknown kinetic constants of apoptotic biochemical reactions proceeding from the condition that the optimal concentrations of procaspases resulting from the solution of the direct optimization problem coincide with the observed ones, that is, those determined by biochemical methods. The Multidimensional Index Method was used to perform numerical solution of the inverse optimization problem.     

A simple method to engineer a protein-derived redox cofactor for catalysis

The 6 ×-Histidine tag which is commonly used for purification of recombinant proteins was converted to a catalytic redox-active center by incorporation of Co^2 +. Two examples of the biological activity of this engineered protein-derived cofactor are presented. After inactivation of the natural diheme cofactor of MauG, it was shown that the Co^2 +-loaded 6 × His-tag could substitute for the hemes in the H₂O₂-driven catalysis of tryptophan tryptophylquinone biosynthesis. To further demonstrate that the Co^2 +-loaded 6 × His-tag could mediate long range electron transfer, it was shown that addition of H₂O₂ to the Co^2 +-loaded 6 × His-tagged Cu^1 + amicyanin oxidizes the copper site which is 20 Å away. These results provide proof of principle for this simple method by which to introduce a catalytic redox-active site into proteins for potential applications in research and biotechnology.     

Fine structural properties of natural and synthetic glycogens

Glycogen, highly branched (1→4)(1→6)-linked α-d-glucan, can be extracted from natural sources such as animal tissues or shellfish (natural source glycogen, NSG). Glycogen can also be synthesized in vitro from glucose-1-phosphate using the cooperative action of α-glucan phosphorylase (GP, EC 2.4.1.1) and branching enzyme (BE, EC 2.4.1.18), or from short-chain amylose by the cooperative action of BE and amylomaltase (AM, EC 2.4.1.25). It has been shown that enzymatically synthesized glycogen (ESG) has structural and physicochemical properties similar to those of NSG. In this study, the fine structures of ESG and NSG were analyzed using isoamylase and α-amylase. Isoamylase completely hydrolyzed the α-1,6 linkages of ESG and NSG. The unit-chain distribution (distribution of degrees of polymerization (DP) of α-1,4 linked chains) of ESG was slightly narrower than that of NSG. α-Amylase treatment revealed that initial profiles of hydrolyses of ESG and NSG were almost the same: both glycogens were digested slowly, compared with starch. The final products from NSG by α-amylase hydrolysis were glucose, maltose, maltotriose, branched oligosaccharides with DP ? 4, and highly branched macrodextrin molecules with molecular weights of up to 10,000. When ESG was digested with excess amounts of α-amylase, much larger macrodextrins (molecular weight > 10⁶) were detected. In contrast, oligosaccharides with DP 4-7 could not be detected from ESG. These results suggest that the α-1,6 linkages in ESG molecules are more regularly distributed than those in NSG molecules.     

Recent advancements in mass spectrometry–based tools to investigate newly synthesized proteins

Tight regulation of protein translation drives the proteome to undergo changes under influence of extracellular or intracellular signals. Despite mass spectrometry–based proteomics being an excellent method to study differences in protein abundance in complex proteomes, analyzing minute or rapid changes in protein synthesis and abundance remains challenging. Therefore, several dedicated techniques to directly detect and quantify newly synthesized proteins have been developed, notably puromycin-based, bio-orthogonal noncanonical amino acid tagging–based, and stable isotope labeling by amino acids in cell culture–based methods, combined with mass spectrometry. These techniques have enabled the investigation of perturbations, stress, or stimuli on protein synthesis. Improvements of these methods are still necessary to overcome various remaining limitations. Recent improvements include enhanced enrichment approaches and combinations with various stable isotope labeling techniques, which allow for more accurate analysis and comparison between conditions on shorter timeframes and in more challenging systems. Here, we aim to review the current state in this field.     

Efficient elicitation of ginsenoside biosynthesis in cell cultures ofPanax notoginseng by using self-chemically-synthesized jasmonates

A series of fluorine and hydroxyl containing jasmonate derivatives, which were chemically synthesized in our institute, were investigated for their effects on the biosynthesis and heterogeneity of ginsenosides in suspension cultures ofPanax notoginseng cells. Compared to the control (without addition of elicitors), 100 μM of each of the jasmonate was added on day 4 to the suspension cultures ofP. notoginseng cells. It was observed that, jasmonates greatly enhanced the ginsenoside content and the ratio of Rb group to Rg group (i.e. (Rb₁+Rd)/(Rg₁+Re)) in theP. notoginseng cells. Some of the synthetic jasmonates, such as pentafluoropropyl jasmonate (PFPJA), 2-hydroxyethyl jasmonate (HEJA) and 2-hydroxyethoxyethyl jasmonate (HEEJA), could promote the ginsenoside content to 2.55±0.11, 3.65±0.13 and 2.94±0.06 mg/100 mg DW, respectively, compared to that of 0.64±0.06 mg/100 mg DW for the control and 2.17±0.04 mg/100 mg DW by the commercially available methyl jasmonate (MJA); and they could change the respective Rb: Rg ratio to 1.60±0.04, 1.87±0.01 and 1.56±0.05, compared to that of 0.47±0.01 for the control and 1.42±0.06 by MJA. The results suggest that suitable esterification of MJA with fluorine or hydroxyl group could increase the elicitation activity to induce plant secondary metabolism. The information obtained from this study is useful for hyper-production of heterogeneous products by plant cell cultures.     

Restriction map of the region surrounding the EcoRI site in the pCR1 plasmid and analysis of an inserted ovalbumin gene.

We have determined a restriction map of a 1650 base pair region surrounding the EcoRI site of the bacterial plasmid, pCR1. We have used pCR1 as a vector in cloning synthetic ovalbumin double-stranded cDNA. Using the pCR1 restriction map, we have characterized the ovalbumin sequences inserted in one recombinant plasmid, pOvE12. POvE12 appears to contain all, or nearly all, of the sequences found in full length, double-stranded cDNA synthesized in vitro.     

Discovery of new potential triplet acting inhibitor for Alzheimer’s disease via X-ray crystallography,molecular docking and molecular dynamics

Abstract

The most common brain disorder of late life is Alzheimer’s disease (AD), which is highly complicating dementia. There are several drug targets which are reported to control the severe level of AD; notably, acetylcholinesterase, β-Secretase and glycogen synthase kinase enzymes are approached as a good drug targets for AD. Hence, the present study mainly focused to discover newly synthesized molecule (7-propyl-6H-pyrano[3,2-c:5,6-c']dichromene-6,8(7H)-dione) as a potential triplet acting drug for above said enzymes through the analysis of X-ray crystallography, molecular docking, molecular dynamics and quantum chemical calculation. The target drug molecule was crystallized in the monoclinic crystal structure with P21/n space group. The structure was solved by SHELXS and refined by SHELXL. The crystal packing is stabilized by C???H···O type of interactions. Further, the induced fit docking shows that the molecule has high docking score, glide energy, favorable hydrogen bonding and hydrophobic interactions on the protein targets. The molecular dynamics simulation was performed to understand the stability of the molecule in the presence of active site environment. Finally, quantum chemical calculation has been carried out for the molecule in gas phase and for the corresponding molecule lifted from the active site region. The structural comparison between gas phase and active site helps to understand the conformational modification of the molecule in the active site.

Communicated by Ramaswamy H. Sarma