Quantitation of Rat Dopamine Transporter mRNA: Effects of Cocaine Treatment and Withdrawal

Dopamine transporter mRNA levels in the rat substantia nigra were quantified using a sensitive nuclease protection assay with a highly homologous human dopamine transporter cDNA clone. The same probe was also used to visualize dopamine transporter mRNA in the substantia nigra by in situ hybridization. Repeated cocaine administration (15 mg/kg, twice a day for 6.5 days) resulted in a greater than 40% decrease in nigral dopamine transporter mRNA levels. In contrast, dopamine transporter mRNA levels were unchanged after either acute treatment (4 h before death) or repeated cocaine treatment followed by a 72-h withdrawal period. Thus, blockade of the dopamine transporter by repeated cocaine administration may result in the down-regulation of dopamine transporter gene expression in dopamine neurons.     

The determination of the pKa of histidine residues in proteins by Raman difference spectroscopy

Sensitive Raman difference spectroscopy was used to monitor the protonation and deprotonation of histidine residues in apo-transferrin. We have shown previously that the behavior of small molecules and/or small molecular groups bound to proteins or other large macromolecules can be studied by Raman difference spectroscopy (Yue, K.T. et al. (1989) J. Raman Spectrosc. 20, 541-545). Using this method, we have measured the Raman difference spectra of human transferrin at different pH values with respect to pH 8.9, titrating its various histidine residues. About 12 +/- 2 of the 19 residues were titrated. The pH difference spectrum of transferrin obtained is very similar to that of histidine in solution, but with clear differences in the 1200-1400 cm-1 region. A titration curve with pKa of 6.08 +/- 0.01 fit the data of histidine in solution and a value of 6.56 +/- 0.02 was found for the average value of the 12 histidine residues inside transferrin. The technique has enough sensitivity at present to monitor a single histidine residue in a 130 kDa molecule and to determine the titration curve of one residue in a 40 kDa protein.     

时域—频域结合分析法—一种分析果蝇求爱歌的新方法

我们设计了一种时域-频域结合分析法,并用此方法分析了6个种群12种果蝇的求爱歌,发现如果将时域与频域的研究结合起来,对求爱歌进行频谱分析,可以定量地揭示出求爱歌的频域特性及其在时域上的细微变化。我们还对果蝇求爱歌的时域模式进行了初步的探讨,发现它们是在同一基本成分上进行调制而产生的,亲缘关系较近的种具有相近的调制方式。在对杂交后代的求爱歌的频谱分析中,我们还发现频谱上的某些特点是能够遗传的。这一新的研究方法为果蝇的进化遗传学和神经遗传的研究提供了一种新的手段。     

氨基酸酯水解酶产生菌的筛选及其合成头孢立新的研究

From ten genera and 146 bacterial strains, 22 strains producing alpha-amino acid ester hydrolase were selected. Among them, AS 1.586 and 41-2 were the best. The optimal conditions for synthesis of cephalexin by pseudomonas aeruginosa 1.204 were investigated. The optimal pH and temperature for enzymatic synthesis reaction was pH 6.8 and 25 degrees C, respectively. By using 1% 7-ADCA, 3% PGME and 4% biomass, about 70% of 7-ADCA was converted to cephalexin under the mentioned conditions.     

Urinary excretion of a novel hexasaccharide and glycopeptide analogue in fucosidosis.

Repeated Biogel P6 chromatography of the urine from a patient with fucosidosis yielded several fractions containing fucosyloligosaccharides and glycopeptides. Two of these were shown by ¹H nuclear magnetic resonance (¹H-n.m.r.) spectroscopy and permethylation analysis to have the following structures respectively: (I) αfuc (1→3) [βgal (1→4)] βglcNAc (1→2) αman ($\text{1→}\text{3}\text{6}$) βman (1→4) glcNAc and (II) αfuc (1→3) [βgal (1→4)] βglcNAc (1→2) αman ($\text{1→}\text{3}\text{6}$) βman (1→4) βglcNAc (1→4) [αfuc ($\text{1→}\text{3}\text{6}$)] βglcNAc-Asn.     

Intravital lipid droplet labeling and imaging reveals the phenotypes and functions of individual macrophages in vivo

Macrophages play pivotal roles in the maintenance of tissue homeostasis. However, the reactivation of macrophages toward proinflammatory states correlates with a plethora of inflammatory diseases, including atherosclerosis, obesity, neurodegeneration, and bone marrow (BM) failure syndromes. The lack of methods to reveal macrophage phenotype and function in vivo impedes the translational research of these diseases. Here, we found that proinflammatory macrophages accumulate intracellular lipid droplets (LDs) relative to resting or noninflammatory macrophages both in vitro and in vivo, indicating that LD accumulation serves as a structural biomarker for macrophage phenotyping. To realize the staining and imaging of macrophage LDs in vivo, we developed a fluorescent fatty acid analog-loaded poly(lactic-co-glycolic acid) nanoparticle to label macrophages in mice with high efficiency and specificity. Using these novel nanoparticles, we achieved in situ functional identification of single macrophages in BM, liver, lung, and adipose tissues under conditions of acute or chronic inflammation. Moreover, with this intravital imaging platform, we further realized in vivo phenotyping of individual macrophages in the calvarial BM of mice under systemic inflammation. In conclusion, we established an efficient in vivo LD labeling and imaging system for single macrophage phenotyping, which will aid in the development of diagnostics and therapeutic monitoring. Moreover, this method also provides new avenues for the study of lipid trafficking and dynamics in vivo.Supplementary key words: macrophage, inflammation, lipid droplet, nanoparticle delivery, in vivo imaging, fatty acid analog, bone marrow, systemic inflammation, lipid trafficking, biomarker

Macrophages, a type of immune cells, almost reside in all tissues of body, from the skin to the bone marrow (BM) (1). Macrophages have remarkable plasticity, and they can be activated into specific subtypes by modifying their physiology and functions in response to local environmental cues. Activated macrophages are commonly divided into proinflammatory killing subtype and anti-inflammatory repairing subtype. Proinflammatory macrophages responding to bacteria, IFN-γ, and lipopolysaccharide (LPS) are involved in host defense and inflammation, whereas anti-inflammatory macrophages responding to interleukin-4 (IL-4), IL-10, and IL-13 play a pivotal role in tissue homeostasis and remodeling (2). Increasing evidence indicates that the reactivation of macrophages toward proinflammatory states under diverse kinds of stress is correlated with a plethora of inflammatory diseases, such as atherosclerosis, diabetes, obesity, rheumatoid arthritis, neurodegeneration, and BM failure syndromes (3, 4). Thus, characterization of macrophage activation status and the underlying molecular mechanism in situ will help elucidate their functions in these diseases; however, in vivo analysis of the macrophage activation status in their native multicellular microenvironment is challenging.Although lipid droplets (LDs) have been initially described as intracellular fat storage organelles in adipocytes, increasing studies indicate that myeloid cells also form LDs under inflammation and stress (5, 6). Macrophages, as the effector cells of innate immunity, are found to form LDs to support their host defense when exposed to pathogens, such as parasites, bacteria, and viruses (7, 8, 9, 10, 11). However, abnormal LD accumulation in tissue-resident macrophages correlates with the pathogenesis of various inflammatory diseases. For instance, foam cells in atherosclerotic lesions can maintain the local inflammatory response by secreting proinflammatory cytokines (12, 13, 14). Moreover, LD-accumulating microglia contribute to neurodegeneration by producing high levels of reactive oxygen species (ROS) and secreting proinflammatory cytokines (15). These findings indicate that LD accumulation might be a hallmark of macrophages with proinflammatory functions.In this study, based on the typical activation of in vitro BM-derived macrophages, we find that proinflammatory M_{(LPS + IFN-γ)} macrophages are characterized by LD accumulation, whereas resting macrophages and anti-inflammatory M_(IL-4) and M_(IL-10) macrophages do not contain any LDs. These features also hold for Matrigel plug-recruited macrophages and tissue-resident macrophages in mice. These findings demonstrate that LD accumulation could serve as a morphological index to distinguish proinflammatory macrophages from others.It is feasible to distinguish LD-containing cells using imaging techniques, which has translational potential for identification of proinflammatory macrophages in vivo. However, current techniques for LD visualization are traditional in vitro staining method, and in vivo staining and imaging of LD in individual macrophages remains a challenge. Through nanocarrier screening, we selected the poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) as nanocarrier to deliver the lipophilic carbocyanine dye (DiI_C18(5) solid (1,1''-dioctadecyl-3,3,3'',3''-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt) [DiD]) and lipid staining dye (C1-BODIPY 500/510-C12) into macrophages. Using these dual fluorescence-labeled PLGA NPs, we achieved in situ and in vivo functional identification of single macrophages in various tissues under systemic or local inflammatory stress. Collectively, this study establishes an efficient in vivo labeling and imaging system of intracellular LDs for phenotyping the activation status and functions of individual macrophages in their dynamic niche, which is pivotal for disease diagnosis and preclinical research.     

Chromosome-level genome assembly and characterization of Sophora Japonica

Sophora japonica is a medium-size deciduous tree belonging to Leguminosae family and famous for its high ecological, economic and medicinal value. Here, we reveal a draft genome of S. japonica, which was ∼511.49 Mb long (contig N50 size of 17.34 Mb) based on Illumina, Nanopore and Hi-C data. We reliably assembled 110 contigs into 14 chromosomes, representing 91.62% of the total genome, with an improved N50 size of 31.32 Mb based on Hi-C data. Further investigation identified 271.76 Mb (53.13%) of repetitive sequences and 31,000 protein-coding genes, of which 30,721 (99.1%) were functionally annotated. Phylogenetic analysis indicates that S. japonica separated from Arabidopsis thaliana and Glycine max ∼107.53 and 61.24 million years ago, respectively. We detected evidence of species-specific and common-legume whole-genome duplication events in S. japonica. We further found that multiple TF families (e.g. BBX and PAL) have expanded in S. japonica, which might have led to its enhanced tolerance to abiotic stress. In addition, S. japonica harbours more genes involved in the lignin and cellulose biosynthesis pathways than the other two species. Finally, population genomic analyses revealed no obvious differentiation among geographical groups and the effective population size continuously declined since 2 Ma. Our genomic data provide a powerful comparative framework to study the adaptation, evolution and active ingredients biosynthesis in S. japonica. More importantly, our high-quality S. japonica genome is important for elucidating the biosynthesis of its main bioactive components, and improving its production and/or processing.     

西藏草地植物功能性状与多项生态系统服务关系

针对植被功能性状与生态系统服务功能之间的相互关系,构建了西藏草地株高和可食性两种功能性状的9项指标,并基于土壤和植物采样,分析了9项植物功能性状指标和5项生态系统服务指标间的相关性,探讨了4种机制(Mass ratio,Selection,Niche complementarity及Insurance)在西藏草地的适用性。结果表明,9项功能性状指标中,株高Rao和可食种与所有种株高CWM比分别与土壤有机碳、土壤全氮和土壤含水率3项生态系统服务指标呈显著负相关及显著正相关。说明群落植被对光能竞争的互补性及可食性状植株在群落中的光能资源相对竞争力,与土壤固碳、肥力供给及水源涵养有显著相关关系。而群落可食种、优势种、优势种与次优势种对光能资源竞争力水平,可食植株多样性、可食植株在群落中的优势度及其光能资源竞争力均值,对草地生态系统服务无显著影响。西藏草地植物功能性状对多项生态系统服务的影响机制从光能资源竞争角度更符合Niche complementarity和Insurance理论,而从可食功能性状角度更符合Mass ratio和Selection理论。