Creation of a HEK293 cell line stably expressing the proteasome subunit PSMD14 fused with fluorescent protein EGFP and HTBT tag

In the present work, a stable cell line based on human embryonic kidney HEK293 cells, which expresses the proteasome subunit PSMD14 fused to the fluorescent protein EGFP and HTBH tag, has been selected. This protein, PSMD14-EGFP-HTBH, was shown to be completely incorporated into proteasomes, and such complexes have a chymotrypsin-like peptidase activity that is fundamental for proteasomes. The constructed and selected cell line can be used for further fluorescent studies of localization of proteasomes in the cell.     

A new murine osteoblastic cell line immortalized with the SV40 large T antigen

The murine preosteoblastic cell line, MC3T3-E1, is widely used to study bone formation and differentiation in vitro. However, this cell line is unstable in culture. The current study was designed to establish a stable osteoblastic cell line. A mammalian expression vector carrying the SV 40 large T antigen was introduced into a primary culture of cells isolated from the calvaria of newborn mice. Among isolated cell lines, the MN16 cell line was selected for further characterization. The MN16 cell line was cultured for 28 days, and compared with the MC3T3-E1 cell line with or without induction. The expression of bone-related genes was examined using the real-time RT-PCR technique. Alizarin red and von Kossa staining were used to detect mineralization of nodules in the cultures. The cell line showed the characteristics of osteoblastic cells in term of gene expression patterns of various molecular markers and calcium deposition in the cell layer after induction. Furthermore, the MN16 cells showed strong adhesion to the basic domain of collagen, a result that is specific for bone-derived cells. The MN16 cell line was found to be stably differentiated into bone formation cells in vitro and should be useful for studying bone biology.     

Generation and characterization of a novel human IgG1 antibody against vascular endothelial growth factor receptor 2

VEGF and its receptors, especially VEGFR2 (KDR), are known to play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies. This study was aimed at developing a fully human IgG1 antibody (mAb-04) constructed from a phage-derived scFv, targeting the VEGF/VEGFR2 pathway. Firstly, an innovative transfection system, containing two recombinant expression vectors (pMH3 and pCApuro), were introduced into CHO-s cells and clones with higher yield selected accordingly. After an optimal fermentation condition was determined, fed-batch fermentation was performed in 5-L bioreactor with a final yield up to 60 mg/L. Further, cell proliferation, wound healing, transwell invasion, tube formation and chick embryo chorioallantoic membrane assays showed significant anti-angiogenic activity of mAb-04 in vitro and in vivo. In addition, the results of Western blotting indicated the ability of mAb-04 to inhibit VEGF-induced VEGFR2 signaling pathway. Finally, ADCC assay demonstrated that mAb-04 is capable of mediating tumor cell killing in presence of effector cells. This study has therefore proved that the full-length antibody targeting human VEGFR2 has potential clinical applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.     

Solid-phase synthesis of ragweed allergen Ra5

A two-stage strategy was used to synthesize the 45-residue, four-disulfide-bonded ragweed protein allergen Ra5: (i) the protected linear chain was prepared by solid-phase, stepwise synthesis according to the sequence of the fully reduced protein; (ii) after removal from the resin and deprotection by hydrogen fluoride, the reduced chain was purified by gel filtration on Sephadex G-50 and allowed to fold “spontaneously” in the presence of oxidized dithiothreitol to form the disulfide links. The product (synthetic Ra5) was purified to disc-electrophoretic homogeneity by cycling through Sephadex G-50 and CM-Sephadex (overall yield: 6% theoretical). Synthetic Ra5 was closely similar in structure to the natural protein by amino acid analysis. polyacrylamide gel electrophoresis, tryptic peptide mapping, and circular dichroism spectra, as well asin vitro andin vivo immunoassays of antigenic/allergenic activities.     

Economic botany: Past and future

Economic botany, like botany, had its origin with the herbalists. Its development was encouraged by various European rulers during the Age of Exploration and Discovery in the 16th century. Recent developments in the United States include A. F. Hill’s textbook, Economic Botany, in 1937 the founding of the journal Economic Botany by E. H. Fulling in 1947 and the formation of the Society for Economic Botany in 1959 Some of the breakthroughs in the development of agriculture, beginning with its origin some 10,000 yr ago, are given. It is concluded that we now need another significant breakthrough to feed the world’s population adequately in the year 2000 Some look to genetic engineering to provide such a breakthrough.     

Glucan Yield from Enzymatic Hydrolysis of Big Bluestem as Affected by Ecotype and Planting Location Along the Precipitation Gradient of the Great Plains

Three big bluestem (Andropogon gerardii Vitman) ecotypes from central Kansas (Cedar Bluffs (CDB) and Webster (WEB) populations), eastern Kansas (Konza (KON) and Top of the World (TOW) populations), and Illinois (12Mile (12M) and Fults (FUL) populations), as well as the Kaw cultivar, were harvested from four reciprocal garden planting locations (Colby, Hays, and Manhattan, KS, and Carbondale, IL) and used to study effects of ecotype and planting location on glucan content and glucan yield from enzymatic hydrolysis along the Great Plains precipitation gradient (～1,200 to 400 mm mean annual precipitation). The populations varied widely in glucan content (31.8–36.5 %), mass recovery (52.0–59.7 %), and glucan recovery (79.0–87.50 %) after acid treatment, efficiency of enzymatic hydrolysis (EEH) (84.6–88.9 %), and glucan mass yield (20.8–29.3 %). Planting location had significant effects on all variables evaluated except EEH. Ecotype had significant effects on glucan recovery, EEH, and glucan mass yield. In addition, interaction between ecotype and planting location also had significant effects on glucan content and glucan mass yield after enzymatic hydrolysis. Planting location had a stronger influence than ecotype and interaction between location and ecotype. Total glucan mass yield of big bluestem (regardless of ecotype) increased as the Great Plains precipitation gradient increased from west to east. Annual precipitation, growing degree days, and potential evapotranspiration in 2010 accounted for 90, 85, and 78 % of the variation in glucan mass yield.     

Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization

A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC–ESI–MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performed using l-norvaline as standard. A limit of detection as low as 1 fmol/µl with a linear range of up to 125 pmol/µl could be obtained. Intraday and interday precisions were lower than 10 % relative standard deviations for most of the amino acids. Quantification using l-norvaline as internal standard gave very similar results compared to the quantification using deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm).     

Vitamin E and selenium supplementation in geriatric patients

Sodium selenate (8 mg/d), organic selenium (50 μg/d) andd-alpha-tocopherol acetate (400 mg/d) were administered for 1 yr to 15 geriatric patients. Fifteen comparable controls received placebo. The mean age of both groups was 76 yr. The patients were assessed every 2 mo independently by two nurses using the Sandoz Clinical Assessment Geriatric-scale (SCAG). The propositae, showed significant improvement in the following SCAG parameters: depression, anxiety, self-care, mental alertness, emotional lability, motivation and initiative, hostility, interest in the environment, fatigue, anorexia, and general impression. A distinct improvement of the general condition was noticed after only 2 mo and the improvement continued up to the end of the 1-yr study period. The high selenium and vitamin E doses were well tolerated; there were no side effects whatsoever.     

Associations of platelet-activating factor acetylhydrolase (PAF-AH) gene polymorphisms with circulating PAF-AH levels and risk of coronary heart disease or blood stasis syndrome in the Chinese Han population

The circulating level of platelet-activating factor acetylhydrolase (PAF-AH) is a novel biomarker to predict the presence of coronary heart disease. PAF-AH gene polymorphisms may be responsible for the variance of circulating PAF-AH levels in individuals. However, the association of PAF-AH gene polymorphisms with circulating PAF-AH levels and the susceptibility to coronary heart disease (CHD) remains unsolved. Blood stasis syndrome (BSS) of CHD is the most common type of TCM syndromes, and a previous study discovered its relationship with the elevated circulating PAF-AH levels. However, the association of gene polymorphisms and CHD with BSS is unclear at present. In this study, four polymorphisms (R92H, I198T, A379V, V279F) of the PAF-AH gene were genotyped in 570 CHD patients, of which 299 had BSS. In addition, 317 unaffected individuals from the same hospitals served as controls. Plasma PAF-AH levels were measured in 155 controls and 271 CHD patients selected randomly, including 139 CHD patients with BSS. In the Chinese Han population, plasma PAF-AH levels in CHD patients with BSS or without BSS were significantly higher (12.9 ± 6.5 and 11.1 ± 5.0 μM, respectively) than in controls (9.3 ± 5.2 μM); this difference still remained significant after adjustment for traditional risk factors or the inflammatory factors. The R92H polymorphism was highly related to the plasma PAF-AH levels and the risk of CHD, especially among patients with BSS, even with the adjustment for the effects of traditional factors. The I198T polymorphism was highly associated with risk of CHD with BSS, but was associated with neither the risk of CHD with no BSS nor with elevated plasma PAF-AH levels.     

Enhanced production of valerenic acid in hairy root culture of Valeriana officinalis by elicitation

Valerenic acid (VA) is a pharmacologically-active sesquiterpene found in valerian (Valeriana officinalis L., Valerianaceae) roots and rhizomes. The plant produces only small amounts of this metabolite naturally. So, induction of hairy roots as well as elicitation can be useful to increase its commercial production. In this study, Wild-type strain ‘A13’ of Agrobacterium rhizogenes was used to induce hairy roots in valerian. The influence of three different elicitors including Fusarium graminearum extract (FE), methyl jasmonate (MJ) and salicylic acid (SA) on VA production in the selected hairy root line ‘LeVa-C4’ was also investigated. The 23-day-old cultures were treated with different concentrations of the elicitors at exposure time of 3 and 7 days. FE (1%) and MJ (100 µM L^?1) highly promoted VA production at 7 days after elicitation, to a level of 12.31- and 6-fold higher than that of non-elicited controls, respectively, and FE did not exert any negative effects on biomass yield of hairy root. SA did not significantly increase the production of VA. This is the first time study to assess the elicitation of hairy root cultures to promote VA biosynthesis in valerian and the resulting experiments demonstrated that F. graminearum extract and MJ were indeed a potent inducer of VA biosynthesis.     

FastMPJ: a scalable and efficient Java message-passing library

The performance and scalability of communications are key for high performance computing (HPC) applications in the current multi-core era. Despite the significant benefits (e.g., productivity, portability, multithreading) of Java for parallel programming, its poor communications support has hindered its adoption in the HPC community. This paper presents FastMPJ, an efficient message-passing in Java (MPJ) library, boosting Java for HPC by: (1) providing high-performance shared memory communications using Java threads; (2) taking full advantage of high-speed cluster networks (e.g., InfiniBand) to provide low-latency and high bandwidth communications; (3) including a scalable collective library with topology aware primitives, automatically selected at runtime; (4) avoiding Java data buffering overheads through zero-copy protocols; and (5) implementing the most widely extended MPI-like Java bindings for a highly productive development. The comprehensive performance evaluation on representative testbeds (InfiniBand, 10 Gigabit Ethernet, Myrinet, and shared memory systems) has shown that FastMPJ communication primitives rival native MPI implementations, significantly improving the efficiency and scalability of Java HPC parallel applications.     

Expression of genes in the 111.35–116.16 million bp fragment of chromosome 13 in brain of mice with different predisposition to hereditary catalepsy

Catalepsy is a pathological animal behavior that is usually associated with dysfunctions in the striatal pallidal system of the brain and can be caused by different reasons. It was previously demonstrated that hereditary catalepsy is linked to the 111.35–116.16 million bp fragment of chromosome 13 in mice. The level of mRNA content in 42 genes localized in this fragment was determined in the study. Two brain departments that are functionally associated with catalepsy (striatum and substantia nigra) were studied in mice from AKR line (resistant to catalepsy), cataleptic CBA line, and recombinant cataleptic AKR.CBA-D13Mit76 (D13) line. The latter was obtained by the transfer of indicated fragment of chromosome 13 from the CBA line to the genome of the AKR line. It was found that two genes (Ndufs4 and Ppap2a) in the striatum and ten genes (Esm1, Fst, Gm10735, Gm15322, Gm15323, Gm15324, Gm15325, Il6st, Il31ra, and Itga1) in the substantia nigra differ in the level of mRNA expression in AKR and D13 lines. The Mcidas gene mRNA level is lower in both structures in D13 line mice than in the AKR line. The expression of the Hspb3 and Mocs2 genes (that encode heat shock protein and molybdenum cofactor synthesis, respectively) is lower in the substantia nigra of CBA and D13 cataleptic line mice than in the AKR line resistant to catalepsy. These genes are considered to be the most likely candidate genes of the catalepsy. The coexpression of a large amount of genes in these brain structures in sick animals indicates the existence of a complex gene network that regulates hereditary catalepsy.     

Purification of a recombinant histidine-tagged lactate dehydrogenase from the malaria parasite,Plasmodium vivax,and characterization of its properties

Lactate dehydrogenase (LDH) of the malaria parasite, Plasmodium vivax (Pv), serves as a drug target and immunodiagnostic marker. The LDH cDNA generated from total RNA of a clinical isolate of the parasite was cloned into pRSETA plasmid. Recombinant his-tagged PvLDH was over-expressed in E. coli Rosetta2DE3pLysS and purified using Ni²⁺-NTA resin giving a yield of 25–30 mg/litre bacterial culture. The recombinant protein was enzymatically active and its catalytic efficiency for pyruvate was 5.4 × 10⁸ min^?1 M^?1, 14.5 fold higher than a low yield preparation reported earlier to obtain PvLDH crystal structure. The enzyme activity was inhibited by gossypol and sodium oxamate. The recombinant PvLDH was reactive in lateral flow immunochromatographic assays detecting pan- and vivax-specific LDH. The soluble recombinant PvLDH purified using heterologous expression system can facilitate the generation of vivax LDH-specific monoclonals and the screening of chemical compound libraries for PvLDH inhibitors.     

Effect of polymorphisms in the CSN3 (κ-casein) gene on milk production traits in Chinese Holstein Cattle

This study was designed to evaluate significant associations between single nucleotide polymorphisms (SNPs) and milk composition and milk production traits in Chinese Holstein cows. Six SNPs were identified in the κ-casein gene using pooled DNA sequencing. The identified SNPs were genotyped by Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) methods from 507 individuals. Out of six, we identified three non-synonymous SNPs (g.10888T>C, g.10924C>A and g.10944A>G) that changed in the protein product. SIFT (Sorting_Intolerant_From_Tolerant) prediction score (0.01) demonstrated that protein changed Isoleucine > Threonine (g.10888T>C) will affect the phenotypes. Significant associations between identified SNPs and three yield traits (milk, protein and fat) and two composition traits (fat and protein percentages) were found whereas it did not reach significance for fat percentage in haplotypes association. Importantly, the significant SNPs in our results showed a large proportion of the phenotypic variation of milk protein yield and concentration. Our results suggest that CSN3 is an important candidate gene that influences milk production traits, and identified polymorphisms and haplotypes could be used as a genetic marker in programs of marker-assisted selection for the genetic improvement of milk production traits in dairy cattle.     

Catalytic Pyrolysis of Raw and Thermally Treated Cellulose Using Different Acidic Zeolites

Fast pyrolysis of biomass using zeolite catalyst has shown to be effective in improving aromatic production. This study focuses on aromatic production through catalytic pyrolysis of major biomass constituent i.e., cellulose. Furthermore, cellulose was torrefied to understand torrefaction’s effect on pyrolysis products. The influence of SiO₂/Al₂O₃ ratios of zeolite (ZSM-5) catalyst on aromatic production during pyrolysis of raw and torrefied cellulose was investigated. Results showed that the catalyst acidity played a pivotal role in eliminating anhydro sugars and other oxygenated compounds while producing more aromatics. The maximum aromatic yield (~25 wt%) was obtained when ZSM-5 with the highest acidity (SiO₂/Al₂O₃?=?30) was used, while the lowest yield (7.99 wt%) was obtained when the least acidic catalyst was used (SiO₂/Al₂O₃?=?280) for raw cellulose pyrolysis. Torrefaction process showed to have positive effect on the aromatic production from pyrolysis. There were no aromatics produced from pyrolysis of raw cellulose in the absence of catalyst, whereas significant amount of aromatic compounds were produced from both catalytic and noncatalytic pyrolyses of torrefied cellulose. The aromatic hydrocarbons produced from catalytic pyrolysis of torrefied cellulose were 5 % more than those produced from raw cellulose at the highest temperature and catalyst acidity (SiO₂/Al₂O₃?=?30).     

Differences in arthropods found in flowers versus trapped in plant resins on Haplopappus platylepis Phil. (Asteraceae): Can the plant discriminate between pollinators and herbivores?

Plants produce secondary metabolites related to ecologically relevant processes. These compounds include surface secretions such as latex, mucilage and resins that help plants face abiotic and biotic environmental threats such as drought, nutrient deficiency, extreme temperatures and UV radiation, as well as herbivory, pathogenic microorganisms and other natural enemies. We studied the resinous coating found on involucral bracts of Haplopappus platylepis Phil. (Asteraceae). This plant belongs to a speciose genus widely distributed in South America (Lane and Hartman in Am J Bot 83:356, 1996). H. platylepis is characterized by resinous fragrant leaves. In this species, resins cover the involucral bracts as well as young leaves and are also secreted on reproductive stalks in smaller amounts. We carried out chemical analysis and natural history observations in order to identify whether arthropods caught in inflorescence resin differed from the ones freely visiting floral disks. Regarding bracteal adhesive’s chemistry, we identified a mixture of diterpenoids and flavonoids; these compounds form a dense surface resin layer, especially over inflorescence’s bracts. In relation to associated arthropods, we found a marked difference in the main organisms captured by bracteal resin compared to insects we observed foraging on disk florets; Arthrobracus (Coleoptera: Melyridae) and Linepithema (Hymenoptera: Formicidae) were the predominant insects “trapped in resin”, while Diadasia (Hymenoptera: Apidae) was the most frequent “floral visitor”. We propose that bracteal resin of H. platylepis may function as a selective trap for non-mutualistic insects reaching reproductive structures of this plant and discuss other multiple possible roles for this secretion, including protocarnivory.