Characterization of two components of the 2-naphthoate monooxygenase system from Burkholderia sp. strain JT1500

2-Naphthoate monooxygenase, a two-protein system, encoded by the nmoA and nmoB genes, was heterologously overexpressed in Escherichia coli. The proteins used for functional characterization were purified to over 90% homogeneity by affinity chromatography. The oxidative component EnmoA (47.9 kDa) lacked substrate catalysis capability on its own, and the reductive component EnmoB (33.4 kDa) and its truncated derivate EnmoB(T) (25 kDa) possessed nearly identical independent flavin reductase activities, c. 130 micromol min(-1) mg(-1) of protein. The inframe fusioned protein EnmoB(T)A (65.2 kDa), containing NmoB(T) and NmoA peptides showed a stable 2-naphthoate monooxygenase activity of 1.2 micromol min(-1) mg(-1) of protein. This is the first report on the purification of a fused form of a two-component flavoprotein monooxygenase. In the specificity experiment, FAD and NADH were shown to be preferred cosubstrates for EnmoB and EnmoB(T). All these data suggest that NmoB(T)A is a two-component flavoprotein monooxygenase, consisting of an oxygenase and a reductase component. NmoA is a member of the class D flavoprotein monooxygenase, and NmoB is an independent NADH:Flavin oxidoreductase.     

Expression and bioactivity of recombinant segments of human perforin.

The aim of the study was to prepare an active recombinant human perforin by comparing 5 candidate segments of human perforin. Full-length perforin, MAC1 (28-349 aa), MAC2 (166-369 aa), C-100, and N-60 of human perforin were selected as candidate active segments and designated, respectively, HP1, HP2, HP3, HP4, and HP5. The target genes were amplified by PCR and the products were individually subcloned into pGEM-T. The genes for HP1, HP2, HP3, and HP5 were subcloned into pET-DsbA, whereas pET-41a (+) was used as the expression vector of HP4. The fusion proteins were expressed in Escherichia coli BL21pLysS(DE3) and purified using nickel nitrilotriacetic acid (NTA) agarose affinity chromatography. The hemolysis microassay was used as an activity assay of fusion protein. From this study, we obtained the recombinant plasmids pGEM-T-HP1, -HP2, -HP3, -HP4 and -HP5, consisting of 1600, 960, 600, 300bp, and 180, respectively. From these recombinant plasmids, expression plasmids were successfully constructed and expressed in E. coli BL21pLysS(DE3). The resultant fusion proteins, affinity purified using Ni-NTA, were approximately 80, 58, 45, 44, and 30 kDa, respectively. The recombinant proteins were assayed for activity on hemolysis. HP2 and HP5 were the only recombinant proteins that were active in hemolysis, and the hemolytic function was concentration dependent. These results demonstrate that active recombinant forms of perforin can be synthesized in a prokaryote model. The recombinant N-60 and MAC1 (28-349 aa) of human perforin have the function of forming pores. Our study provides the experimental basis for further investigation on the application of perforin.     

纳洛酮在延髓腹外侧加压区加强P物质引起的升压效应

目的：探讨Ｐ物质（ＳＰ）在延髓腹外侧加压区（ＶＳＭｐ）的升压效应是否与内源性阿片样物质有关。方法：氨基甲酸乙酯麻醉、人工呼吸,暴露延髓腹侧面,ＶＳＭｐ 敷贴ＳＰ等药物。结果：ＶＳＭｐ 敷贴ＳＰ可使血压呈剂量效应关系的明显增高,但心率无明显变化;在ＶＳＭｐ 给纳洛酮进行预处理,可显著增强ＳＰ的升压效应;应用神经元细胞体兴奋剂Ｌ谷氨酸钠到ＶＳＭｐ 可使血压显著升高。结论：ＳＰ 在ＶＳＭｐ 具有明显的升压作用,内源性阿片样物质在ＶＳＭｐ 对ＳＰ升压效应起拮抗作用,ＳＰ的升压效应可能是兴奋ＶＳＭｐ 的神经元细胞体实现的     

Role of asymmetric dimethylarginine in homocysteine-induced apoptosis of vascular smooth muscle cells

Homocysteine (Hcy) could induce apoptosis of vascular smooth muscle cells (VSMC). Asymmetric dimethylarginine (ADMA) has been thought as a novel risk factor for cardiovascular diseases. We hypothesized that ADMA mediates homocysteine-induced apoptosis of VSMC. In this experiment the level of ADMA in the medium measured by high-performance liquid chromatography (HPLC) was elevated when the apoptosis of T/G HA-VSMC was induced by Hcy which was detected by Hoechst33342 staining or flow cytometry (FCM) with Annecin V+Propidium Iodide (PI). Exogenous ADMA induced the apoptosis of VSMC. At the same time, ADMA elevated the level of intracellular reactive oxidative species (ROS) determined by fluorescent ROS detection kit. The activation of JNK and p38MAPK contributed to ADMA-induced apoptosis of VSMC. The present results suggest that endogenous ADMA is involved in apoptosis of VSMC induced by Hcy, and the effects of ADMA is related to elevation of intracellular ROS and activation of JNK/p38MAPK signaling pathways.     

Water and Nutrient Use Efficiency in Diploid, Tetraploid and Hexaploid Wheats

Three diploid (Triticum boeoticum, AA; Aegilops speltoides, BB and Ae. tauschii, DD), two tetraplold (T. dlcoccoides,AABB and T. dicoccon, AABB) and one hexaploid (T. vulgare, AABBDD) varieties of wheat, which are very important in the evolution of wheat were chosen in this study. A pot experiment was carried out on the wheat under different water and nutrient conditions (i) to understand the differences in biomass, yield, water use efficiency (WUE), and nutrient (N, P and K) use efficiency (uptake and utilization efficiency) among ploldles in the evolution of wheat; (ii) to clarify the effect of water and nutrient conditions on water and nutrient use efficiency; and (iii) to assess the relationship of water and nutrient use efficiency in the evolution of wheat. Our results showed that from diploid to tetraploid then to hexaploid during the evolution of wheat, both root biomass and above-ground biomass increased initially and then decreased. Water consumption for transpiration decreased remarkably, correlating with the decline of the growth period, while grain yield, harvest index, WUE, N, P and K uptake efficiency, and N, P and K utilization efficiency increased significantly. Grain yield, harvest index and WUE decreased in the same order: T.vulgare ＞ T. dicoccon ＞ T. dicoccoides ＞ Ae. tauschii ＞ Ae. speltoides ＞ T. boeoticum. Water stress significantly decreased root biomass, above-ground biomass, yield, and water consumption for transpiration by 47-52%, butremarkably increased WUE. Increasing the nutrient supply increased wheat above-ground biomass, grain yield,harvest index, water consumption for transpiration and WUE under different water levels, but reduced root biomass under drought conditions. Generally, water stress and low nutrient supply resulted in the lower nutrientuptake efficiency of wheat. However, water and nutrient application had no significant effects on nutrient utilization efficiency, suggesting that wheat nutrient utilization efficiency is mainly controlled by genotypes. Compared to theother two diploid wheats, Ae. squarrosa (DD) had significant higher WUE and nutrient utilization efficiency, Indicating that the D genome may carry genes controlling high efficient utilization of water and nutrient. Significant relationships were found between WUE and N, P and K utilization efficiency.     

Molecular characterization of <Emphasis Type="Italic">HOXC8</Emphasis> gene and methylation status analysis of its exon 1 associated with the length of cashmere fiber in Liaoning cashmere goat

Homeobox protein Hox-C8 (HOXC8) is a member of Hox family. It is expressed in the dermal papilla of the skin and is thought to be associated with the hair inductive capacity of dermal papilla cells. In the present study, we isolated and characterized a full-length open reading frame of HOXC8 cDNA from the skin tissue of Liaoning cashmere goat, as well as, established a phylogenetic relationship of goat HOXC8 with that of other species. Also, we investigated the effect of methylation status of HOXC8 exon 1 at anagen secondary hair follicle on the cashmere fiber traits in Liaoning cashmere goat. The sequence analysis indicated that the obtained cDNA was 1134-bp in length containing a complete ORF of 729-bp. It encoded a peptide of 242 amino acid residues in length. The structural analysis indicated that goat HOXC8 contained a typical homeobox domain. The phylogenetic analysis revealed that Capra hircus HOXC8 had a closer genetic relationship with that of Ovis aries, followed by Bos Taurus and Bubalus bubalis. The methylation analysis suggested that the methylation degree of HOXC8 exon 1 in anagen secondary hair follicle might be involved in regulating the growth of cashmere fiber in Liaoning cashmere goat. Our results provide new evidence for understanding the molecular structural and evolutionary characteristics of HOXC8 in Liaoning cashmere goat, as well as, for further insight into the role of methylation degree of HOXC8 exon 1 regulates the growth of cashmere fiber in goat.     

Reliable Method for Fabricating Tissue‐Mimicking Materials With Designated Relative Permittivity and Conductivity at 128 MHz

Artificial materials that can simultaneously mimic the relative permittivity and conductivity of various human tissues are usually used in medical applications. However, the method of precisely designing these materials with designated values of both relative permittivity and conductivity at 3 T MRI resonance frequency is lacking. In this study, a reliable method is established to determine the compositions of artificial dielectric materials with designated relative permittivity and conductivity at 128 MHz. Sixty dielectric materials were produced using oil, sodium chloride, gelatin, and deionized water as the main raw materials. The dielectric properties of these dielectric materials were measured using the open‐ended coaxial line method at 128 MHz. Nonlinear least‐squares Marquardt–Levenberg algorithm was used to obtain the formula, establishing the relationship between the compositions of the dielectric materials and their dielectric properties at 128 MHz. The dielectric properties of the blood, gall bladder, muscle, skin, lung, and bone at 128 MHz were selected to verify the reliability of the obtained formula. For the obtained formula, the coefficient of determination and the expanded uncertainties with a coverage factor of k = 2 were 0.991% and 4.9% for relative permittivity and 0.992% and 6.4% for conductivity. For the obtained artificial materials measured using the open‐ended coaxial line method, the maximal difference of relative permittivity and conductivity were 1.0 and 0.02 S/m, respectively, with respect to the designated values. In conclusion, the compositions of tissue‐mimicking material can be quickly determined after the establishment of the formulas with the expanded uncertainties of less than 10%. Bioelectromagnetics. 2021;42:86–94. © 2020 Bioelectromagnetics Society.     

Identification of compound heterozygous DNAH11 variants in a Han-Chinese family with primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a group of genetically and clinically heterogeneous disorders with motile cilia dysfunction. It is clinically characterized by oto-sino-pulmonary diseases and subfertility, and half of the patients have situs inversus (Kartagener syndrome). To identify the genetic cause in a Han-Chinese pedigree, whole-exome sequencing was conducted in the 37-year-old proband, and then, Sanger sequencing was performed on available family members. Minigene splicing assay was applied to verify the impact of the splice-site variant. Compound heterozygous variants including a splice-site variant (c.1974-1G>C, rs1359107415) and a missense variant (c.7787G>A, p.(Arg2596Gln), rs780492669), in the dynein axonemal heavy chain 11 gene (DNAH11) were identified and confirmed as the disease-associated variants of this lineage. The minigene expression in vitro revealed that the c.1974-1G>C variant could cause skipping over exon 12, predicted to result in a truncated protein. This discovery may enlarge the DNAH11 variant spectrum of PCD, promote accurate genetic counselling and contribute to PCD diagnosis.