High-performance liquid chromatography of fatty acid isopropylidene hydrazides and its application in lipid analysis

Fatty acid isopropylidene hydrazides, prepared by stepwise treatment of acyl lipids with hydrazine and acetone, were analyzed by high-performance liquid chromatography on a reversed-phase column. These derivatives could be easily eluted with 15% water in methanol and monitored by measuring absorbance at 229 nm with a uv detector. Their elution behavior, in general, was similar to that of methyl esters and some commonly used ultraviolet-absorbing derivatives of fatty acids. The new method has been used for fatty acid analysis of some oils.     

High-throughput,Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography

The bacterial cell wall is a network of glycan strands cross-linked by short peptides (peptidoglycan); it is responsible for the mechanical integrity of the cell and shape determination. Liquid chromatography can be used to measure the abundance of the muropeptide subunits composing the cell wall. Characteristics such as the degree of cross-linking and average glycan strand length are known to vary across species. However, a systematic comparison among strains of a given species has yet to be undertaken, making it difficult to assess the origins of variability in peptidoglycan composition. We present a protocol for muropeptide analysis using ultra performance liquid chromatography (UPLC) and demonstrate that UPLC achieves resolution comparable with that of HPLC while requiring orders of magnitude less injection volume and a fraction of the elution time. We also developed a software platform to automate the identification and quantification of chromatographic peaks, which we demonstrate has improved accuracy relative to other software. This combined experimental and computational methodology revealed that peptidoglycan composition was approximately maintained across strains from three Gram-negative species despite taxonomical and morphological differences. Peptidoglycan composition and density were maintained after we systematically altered cell size in Escherichia coli using the antibiotic A22, indicating that cell shape is largely decoupled from the biochemistry of peptidoglycan synthesis. High-throughput, sensitive UPLC combined with our automated software for chromatographic analysis will accelerate the discovery of peptidoglycan composition and the molecular mechanisms of cell wall structure determination.     

Hydroxycinnamoyl amides in stem explants from flowering and non-flowering Nicotiana tabacum

Flower formation in stem explants was chosen as an experimental system for the study of the function of hydroxycinnamoyl amides (HCAs) in plants. The explants, derived from flowering and non-flowering Nicotiana tabacum L. var. Xanthi nc., differentiated to two types of callus and afterwards to flower buds. A novel reversed-phase high performance liquid chromatography method, following sample clean-up on CM-Fractogel columns, enabled us to examine HCA concentrations in small tissue samples. Two different groups of HCA could be distinguished during in vitro flower formation: Firstly, feruloyl- and diferuloyputrescine, the major detectable HCAs that accumulated during callus proliferation; secondly, caffeoylputrescine, which accumulated during the later stage of flower differentiation and reached higher concentrations than feruloylputrescine.     

Conversion of iodine to fluorine-18 based on iodinated chalcone and evaluation for β-amyloid PET imaging

In the amyloid cascade hypothesis, β-amyloid (Aβ) plaques is one of the major pathological biomarkers in the Alzheimer’s disease (AD) brain. We report the synthesis and evaluation of novel radiofluorinated chalcones, [¹⁸F]4-dimethylamino-4′-fluoro-chalcone ([¹⁸F]DMFC) and [¹⁸F]4′-fluoro-4-methylamino-chalcone ([¹⁸F]FMC), as Aβ imaging probes. The conversion of iodine directly introduced to the chalcone backbone into fluorine was successfully carried out by ¹⁸F-labeling via the corresponding boronate precursors, achieving the direct introduction of fluorine-18 into the chalcone backbone to prepare [¹⁸F]DMFC and [¹⁸F]FMC. In a biodistribution study using normal mice, [¹⁸F]DMFC and [¹⁸F]FMC showed a higher initial uptake (4.43 and 5.47% ID/g at 2?min postinjection, respectively) into and more rapid clearance (0.52 and 0.66% ID/g at 30?min postinjection, respectively) from the brain than a Food and Drug Administration (FDA)-approved Aβ imaging agent ([¹⁸F]Florbetapir), meaning the improvement of the probability of detecting Aβ plaques and the reduction of non-specific binding in the brain. In the in vitro binding studies using aggregates of recombinant Aβ peptides, [¹⁸F]DMFC and [¹⁸F]FMC showed high binding affinity to recombinant Aβ aggregates at the K_d values of 4.47 and 6.50?nM, respectively. In the in vitro autoradiography (ARG) experiment with AD brain sections, [¹⁸F]DMFC and [¹⁸F]FMC markedly accumulated only in a region with abundant Aβ plaques, indicating that they clearly recognized human Aβ plaques in vitro. These encouraging results suggest that [¹⁸F]DMFC and [¹⁸F]FMC may be promising PET probes for the detection of an amyloid pathology and the early diagnosis of AD with marked accuracy.     

A bidirectional crosstalk between glioblastoma and brain endothelial cells potentiates the angiogenic and proliferative signaling of sphingosine-1-phosphate in the glioblastoma microenvironment

Glioblastoma is one of the most malignant, angiogenic, and incurable tumors in humans. The aberrant communication between glioblastoma cells and tumor microenvironment represents one of the major factors regulating glioblastoma malignancy and angiogenic properties. Emerging evidence implicates sphingosine-1-phosphate signaling in the pathobiology of glioblastoma and angiogenesis, but its role in glioblastoma-endothelial crosstalk remains largely unknown. In this study, we sought to determine whether the crosstalk between glioblastoma cells and brain endothelial cells regulates sphingosine-1-phosphate signaling in the tumor microenvironment. Using human glioblastoma and brain endothelial cell lines, as well as primary brain endothelial cells derived from human glioblastoma, we report that glioblastoma-co-culture promotes the expression, activity, and plasma membrane enrichment of sphingosine kinase 2 in brain endothelial cells, leading to increased cellular level of sphingosine-1-phosphate, and significant potentiation of its secretion. In turn, extracellular sphingosine-1-phosphate stimulates glioblastoma cell proliferation, and brain endothelial cells migration and angiogenesis. We also show that, after co-culture, glioblastoma cells exhibit enhanced expression of S1P₁ and S1P₃, the sphingosine-1-phosphate receptors that are of paramount importance for cell growth and invasivity. Collectively, our results envision glioblastoma-endothelial crosstalk as a multi-compartmental strategy to enforce pro-tumoral sphingosine-1-phosphate signaling in the glioblastoma microenvironment.     

贝叶斯方法估计阈性状育种值的原理和计算技术

本文介绍了估计阈性状育种值的贝叶斯方法的原理,演示了描述阈性状观察值、建立后验概率密度函数、以及导出非线性方程组的方法．并就这一估计方法的计算技术进行了讨论,针对动物遗传育种中方程组系数矩阵往往很大,超出计算机内存的情况,提出了不需要建立方程组,在数据上迭代求解的计算方法．本文还综述了这一非线性方法与线性方法在阈性状育种值估计上的比较．     

Phenotypic and genotypic intra-diversity among Anatolian durum wheat “Kunduru” landraces

Kunduru is an important Anatolian landrace having peculiar traits that are appreciated by farmers and breeders. 33 accessions known as Kunduru collected by ICARDA from six geographical provinces of Turkey, were used to study the phenotypic and genotypic intra-diversity. Kunduru landraces exhibited high intra-diversity for most of the studied morphological traits. GPC (12.10–14.90%), vitreousness (75–100%), TKW (31.80–56.70 g), YP (4.70–8.00 ppm), b*-value (14.30–19.50), ash content (1.60–2.0%) and gluten strength (14–60 ml) showed marked variations. Gliadin and glutenin banding patterns showed high polymorphism. 65 alleles were detected with 14 SSR markers, giving a mean of 6.77 alleles per locus. The average PIC value was 0.44 and ranged from 0.11 to 0.70. The average genetic distance between pairs of landraces was 0.47 and ranged between 0.11 and 0.72. This study showed that Kunduru landraces maintains high allelic variation. PCoA indicated that eco-geographical variables have a significant effect on SSR diversity as well as morphological traits. Many of the landraces studied are in danger of disappearing from the local farmers' fields; this study demonstrates the importance of maintaining and conserving this precious genetic resources.     

Characterization and autoprocessing of precursor and mature forms of human immunodeficiency virus type 1 (HIV 1) protease purified from Escherichia coli

A recombinant plasmid encompassing the human immunodeficiency virus type 1 (HIV 1) protease coding sequence and flanking regions (Ala-13 to Gly-185 of the pol open reading frame) has been expressed in two distinct strains of Escherichia coli, AR58 and AR68. In the first strain, AR58, the primary translation product, a 25 kilodalton (kDa) precursor protein, is short-lived and rapidly processes itself to the 11 kDa mature protease in vivo. In the second strain, AR68, the 25 kDa species is only partially processed, and it, a 13 kDa intermediate, and the mature 11 kDa enzyme accumulate at a ratio of 3:4.5:2.5, respectively. The 11 kDa mature protease from AR58 and the 25 kDa precursor from AR68 have been purified to homogeneity. The yield of 11 kDa enzyme from AR58 is approximately 0.02 mg/g wet weight of E. coli cell pellet. The protease has both the expected NH2- and COOH-terminal sequences. The yield of 25 kDa enzyme from AR68 is approximately 0.1 mg/g wet weight of E. coli cell pellet. In vitro, the 25 kDa precursor enzyme rapidly (t1/2 approximately equal to 9 min) processes itself into a species with a mass of approximately 13 kDa and a species with a mass of approximately 11 kDa. Both of these latter species can be separated by RP-HPLC, have the NH2-terminal sequence expected for the mature protease, and are active. The 11 kDa enzyme from AR58 comigrates with the 11 kDa enzyme from AR68 on RP-HPLC and SDS polyacrylamide gel electrophoresis. On extended incubation at 4 degrees C at either neutral or acidic pH all species of the protein exhibit further autodegradation at defined sequences. The availability of the mature, 11 kDa enzyme and the 25 kDa precursor will allow biochemical and physical studies on this critical viral enzyme.