Mitochondrial oxygen tension within the heart

By using a newly developed optical technique which enables non-invasive measurement of mitochondrial oxygenation (mitoPO₂) in the intact heart, we addressed three long-standing oxygenation questions in cardiac physiology: 1) what is mitoPO₂ within the in vivo heart?, 2) is mitoPO₂ heterogeneously distributed?, and 3) how does mitoPO₂ of the isolated Langendorff-perfused heart compare with that in the in vivo working heart? Following calibration and validation studies of the optical technique in isolated cardiomyocytes, mitochondria and intact hearts, we show that in the in vivo condition mean mitoPO₂ was 35 ± 5 mm Hg. The mitoPO₂ was highly heterogeneous, with the largest fraction (26%) of mitochondria having a mitoPO₂ between 10 and 20 mm Hg, and 10% between 0 and 10 mm Hg. Hypoxic ventilation (10% oxygen) increased the fraction of mitochondria in the 0–10 mm Hg range to 45%, whereas hyperoxic ventilation (100% oxygen) had no major effect on mitoPO₂. For Langendorff-perfused rat hearts, mean mitoPO₂ was 29 ± 5 mm Hg with the largest fraction of mitochondria (30%) having a mitoPO₂ between 0 and 10 mm Hg. Only in the maximally vasodilated condition, did the isolated heart compare with the in vivo heart (11% of mitochondria between 0 and 10 mm Hg). These data indicate 1) that the mean oxygen tension at the level of the mitochondria within the heart in vivo is higher than generally considered, 2) that mitoPO₂ is considerably heterogeneous, and 3) that mitoPO₂ of the classic buffer-perfused Langendorff heart is shifted to lower values as compared to the in vivo heart.     

Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

The structure and dynamics of proteins underlies the workings of virtually every biological process. Existing biophysical methods are inadequate to measure protein structure at atomic resolution, on a rapid time scale, with limited amounts of protein, and in the context of a cell or membrane. FRET can measure distances between two probes, but depends on the orientation of the probes and typically works only over long distances comparable with the size of many proteins. Also, common probes used for FRET can be large and have long, flexible attachment linkers that position dyes far from the protein backbone. Here, we improve and extend a fluorescence method called transition metal ion FRET that uses energy transfer to transition metal ions as a reporter of short-range distances in proteins with little orientation dependence. This method uses a very small cysteine-reactive dye monobromobimane, with virtually no linker, and various transition metal ions bound close to the peptide backbone as the acceptor. We show that, unlike larger fluorophores and longer linkers, this donor–acceptor pair accurately reports short-range distances and changes in backbone distances. We further extend the method by using cysteine-reactive metal chelators, which allow the technique to be used in protein regions of unknown secondary structure or when native metal ion binding sites are present. This improved method overcomes several of the key limitations of classical FRET for intramolecular distance measurements.     

Probing of the rates of alternating access in LacY with Trp fluorescence

Sugar/H⁺ symport by lactose permease (LacY) utilizes an alternating access mechanism in which sugar and H⁺ binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by sequential opening and closing of inward- and outward-facing hydrophilic cavities. Here, we introduce Trp residues on either side of LacY where they are predicted to be in close proximity to side chains of natural Trp quenchers in either the inward- or outward-facing conformers. In the inward-facing conformer, LacY is tightly packed on the periplasmic side, and Trp residues placed at positions 245 (helix VII) or 378 (helix XII) are in close contact with His-35 (helix I) or Lys-42 (helix II), respectively. Sugar binding leads to unquenching of Trp fluorescence in both mutants, a finding clearly consistent with opening of the periplasmic cavity. The pH dependence of Trp-245 unquenching exhibits a pK_a of 8, typical for a His side chain interacting with an aromatic group. As estimated from stopped-flow studies, the rate of sugar-induced opening is ≈100 s⁻¹. On the cytoplasmic side, Phe-140 (helix V) and Phe-334 (helix X) are located on opposite sides of a wide-open hydrophilic cavity. In precisely the opposite fashion from the periplasmic side, mutant Phe-140→Trp/Phe-334→His exhibits sugar-induced Trp quenching. Again, quenching is pH dependent (pK_a = 8), but remarkably, the rate of sugar-induced quenching is only ≈0.4 s⁻¹. The results provide yet another strong, independent line of evidence for the alternating access mechanism and demonstrate that the methodology described provides a sensitive probe to measure rates of conformational change in membrane transport proteins.     

Fluorescence spectroscopy to diagnose hepatic steatosis in a rat model of fatty liver

Background: Steatosis is diagnosed on the basis of the macroscopic aspect of the liver evaluated by the surgeon at the time of organ extraction or by means of a frozen biopsy. Aim: In the present study, the applicability of laser‐induced fluorescence (LIF) spectroscopy was investigated as a method for the diagnosis of different degrees of steatosis experimentally induced in rats. Material and methods: Rats received a high‐lipid diet for different periods of time. The animals were divided into groups according to the degree of induced steatosis diagnosis by histology. The concentration of fat in the liver was correlated with LIF by means of the steatosis fluorescence factor (SFF). Results: The histology classification, according to liver fat concentration was, Severe Steatosis, Moderate Steatosis, Mild Steatosis and Control (no liver steatosis). Fluorescence intensity could be directly correlated with fat content. It was possible to estimate an average of fluorescence intensity variable by means of different confidence intervals (P=95%) for each steatosis group. SFF was significantly higher in the Severe Steatosis group (P<0.001) compared with the Moderate Steatosis, Mild Steatosis and Control groups. Conclusion: The various degrees of steatosis could be directly correlated with SFF. LIF spectroscopy proved to be a method capable of identifying the degree of hepatic steatosis in this animal model, and has the potential of clinical application for non‐invasive evaluation of the degree of steatosis.     

中国人黏膜相关淋巴组织结外边缘区淋巴瘤染色体易位的临床研究

目的 探讨中国人不同部位黏膜相关淋巴组织结外边缘区淋巴瘤(MALTL)中分子遗传学异常的发生情况.方法 应用间期荧光原位杂交(FISH)方法,检测217例不同部位MALTL的t(11;18)(q21;q21)/API2-MALT1、t(1;14)(p22;q32)/IGH-BCL10、t(14;18)(q32;q21)/IGH-MALT1和涉及BCL6基因的染色体易位.结果 染色体易位的总发生率为21%(46/217).全部病例共检测出4种主要染色体异常,其中第1种13%(29/217)为t(11;18)(q21;q21)/API2-MALT1,最常见的发生部位是肺47%(8/17)和小肠29%(4/14),其次为唾液腺1/6例、胃14%(12/84)和眼附属器6%(4/68).第2种1%(3/217)为t(1;14)(p22;q32)/IGH-BCL10,仪见于肺12%(2/17)和胃1%(1/84).第3种1%(2/217)为t(14;18)(q32;q21)/IGH-MALTI,仅见于肺6%(1/17)和眼附属器2%(1/68).第4种2%(4/217)为BCL6基因涉及的染色体易位,见于唾液腺1/6例和胃4%(3/84).4%(8/217)为涉及IGH基因但未知与其易位的伙伴基因的染色体易位.结论 以上4种染色体易位在中国人不同解剖部位的MALTL中的发生率有明显不同,与欧美国家所报道的染色体易位的发生率相比较,分布稍有差异.     

牛黄解毒片中砷元素分析

目的对牛黄解毒片中总砷、可溶性砷、可溶性砷（Ⅲ）和砷（Ⅴ）进行分析,为评价牛黄解毒片的安全性提供依据。方法采用微波消解-原子荧光法测定牛黄解毒片中的总砷含量,50%甲醇浸提-原子荧光法测定可溶性砷,717阴离子交换树脂分离-原子荧光法测定可溶性砷（Ⅲ）和砷（Ⅴ）。结果3种牛黄解毒片制剂中总砷含量为65.72～75.79g/kg,可溶性砷占总砷百分比为0.43%～1.23%,其中可溶性砷（Ⅲ）含量0.19～0.55g/kg,可溶性砷（Ⅴ）含量范围在0.09～0.44g/kg。结论不同的牛黄解毒片中可溶性砷及其中两种价态砷的含量差异较大,因此有必要对牛黄解毒片中砷的含量进行质量控制。     

环孢素治疗药物监测的质量控制

目的考察荧光偏振免疫（FPIA）法测定环孢素A（CsA）血药浓度的可行性及本实验室条件下CsA的治疗药物监测质量控制水平。方法以标准质控为样本,考察FPIA法测定CsA血药浓度的方法学性质,对本实验室2008年质控数据进行统计分析。结果方法低、中、高浓度回收率为92.83%～100.43%,RSD为2.49%～6.12%;本院2008年度随行质控低、中、高浓度RSD分别为5.85%,5.41%,4.95%,符合中国药典要求。质控图基本符合正态分布,存在一定程度的趋势性变化。结论 FPIA法具有良好的准确度和精密度,在长期的检测中稳定性较好,适合临床开展治疗药物监测,但应建立合理质控体系。     

Quantitative analysis of neuropeptide Y receptor association with ��-arrestin2 measured by bimolecular fluorescence complementation

Background and purpose:

β-Arrestins are critical scaffold proteins that shape spatiotemporal signalling from seven transmembrane domain receptors (7TMRs). Here, we study the association between neuropeptide Y (NPY) receptors and β-arrestin2, using bimolecular fluorescence complementation (BiFC) to directly report underlying protein–protein interactions.

Experimental approach:

Y1 receptors were tagged with a C-terminal fragment, Yc, of yellow fluorescent protein (YFP), and β-arrestin2 fused with the complementary N-terminal fragment, Yn. After Y receptor–β-arrestin association, YFP fragment refolding to regenerate fluorescence (BiFC) was examined by confocal microscopy in transfected HEK293 cells. Y receptor/β-arrestin2 BiFC responses were also quantified by automated imaging and granularity analysis.

Key results:

NPY stimulation promoted association between Y1–Yc and β-arrestin2–Yn, and the specific development of BiFC in intracellular compartments, eliminated when using non-interacting receptor and arrestin mutants. Responses developed irreversibly and were slower than for downstream Y1 receptor–YFP internalization, a consequence of delayed maturation and stability of complemented YFP. However, β-arrestin2 BiFC measurements delivered appropriate ligand pharmacology for both Y1 and Y2 receptors, and demonstrated higher affinity of Y1 compared to Y2 receptors for β-arrestin2. Receptor mutagenesis combined with β-arrestin2 BiFC revealed that alternative arrangements of Ser/Thr residues in the Y1 receptor C tail could support β-arrestin2 association, and that Y2 receptor–β-arrestin2 interaction was enhanced by the intracellular loop mutation H155P.

Conclusions and implications:

The BiFC approach quantifies Y receptor ligand pharmacology focused on the β-arrestin2 pathway, and provides insight into mechanisms of β-arrestin2 recruitment by activated and phosphorylated 7TMRs, at the level of protein–protein interaction.     

Improved structural characterization of chromosomal breakpoints using high resolution custom array‐CGH

Lindstrand A, Schoumans J, Gustavsson P, Hanemaaijer N, Malmgren H, Blennow E. Improved structural characterization of chromosomal breakpoints using high resolution custom array‐CGH. Array‐CGH is a powerful tool for the rapid detection of genomic imbalances. By customizing the array it is possible to increase the resolution in a targeted genomic region of interest and determine the structure of the breakpoints with high accuracy, as well as to detect very small imbalances. We have used targeted custom arrays to zoom in on 38 chromosomal breakpoints from 12 different patients carrying both balanced and unbalanced rearrangements. We show that it is possible to characterize unbalanced breakpoints within 17–20,000 bp, depending on the structure of the genome. All of the deletion and duplication breakpoints were further refined and potential underlying molecular mechanisms of formation are discussed. In one of seven carriers of apparently balanced reciprocal translocations we detected a small deletion of 200 bp within the previously FISH‐defined breakpoint, and in another patient, a large deletion of 11 Mb was identified on a chromosome not involved in the translocation. Targeted custom oligonucleotide arrays make it possible to perform fine mapping of breakpoints with a resolution within the breakpoint region much higher compared to commercially available array platforms. In addition, identification of small deletions or duplications in apparently balanced rearrangements may contribute to the identification of new disease causing genes.     

前列腺癌8号染色体改变与Gleason评分之间的相关性

目的探索前列腺癌8号染色体数目增加及c-myc基因和脂蛋白脂酶（LPL）基因状态和发生频率,分析8号染色体改变与前列腺癌Gleason评分之间的相关性及其在前列腺癌发生发展中的作用。方法采用ProVysion^TM三色探针组合,以荧光原位杂交（FISH）方法检测34例未经临床治疗的前列腺癌穿刺组织石蜡切片标本的8号染色体改变,其中包括Gleason评分5分者1例,6分者10例,7分者14例,8分者4例,9分者5例,并进行8号染色体各种异常之间及其与前列腺癌Gleason评分级别之间的关联性分析。结果8号染色体增加为17／34（50％）,c-myc基因拷贝数增加为21／34（61．8％）,LPL单体为15／34（44．1％）,c-myc基因扩增为23／34（67．6％）,LPL基因缺失为21／34（61．8％）,同时具有LPL基因缺失和c-myc基因扩增为16／34（47．1％）,至少有其中一种遗传学异常者为29／34（85．3％）。8号染色体增加与Gleason评分级别增高呈明显的正相关关系（P=0．0006）;c-myc基因拷贝数增加与Gleason评分级别增高呈正相关关系（P=0．0035）;LPL缺失与Gleason评分级别增高呈负相关关系（P=0．0383）;调整年龄后,除了上述三个变量与Gleason评分级别的相关关系仍然存在以外,c-myc基因扩增与Gleason评分级别增高也呈现正相关关系（P=0．0462）。结论8号染色体数目增加、c-myc基因拷贝数增加、c-myc基因扩增和LPL基因丢失都与Gleason评分级别有关,c-myc基因扩增同时伴有LPL基因缺失也是前列腺癌的遗传学特征之一,提示8号染色体异常可能与前列腺癌的发生和进展有关。