Use of a fluorescent dye to measure secretion from intact bovine anterior pituitary cells

The fluorescent dye FM1-43 has been used to indicate membrane changes in individual bovine anterior pituitary cells exposed to secretory stimuli. After ten minutes incubation with FM1-43 (2 M), cells showed three patterns of dye fluorescence: annular, partly filled and uniformly filled. FM1-43 fluorescence was increased in 61% of the cells by TRH (40 nM), a physiological stimulus for prolactin secretion, and in 89% of the cells by 60 mM external K⁺. The fluorescence also increased when cells incubated in the presence of quinpirole, a dopamine D2-receptor agonist which inhibits prolactin secretion, were exposed to raclopride, a D-2 antagonist. The increases in FM1-43 fluorescence caused by these treatments suggests that the dye acts as an indicator of secretion, possibly through incorporation into secretory vesicle membranes exposed on the cell surface during exocytosis. If the dye was washed away after loading, the fluorescence of partly and uniformly filled cells was retained and a rise in fluorescence could still be seen on stimulation by TRH. This suggests that some dye had been taken up by endocytosis and trapped in an intracellular compartment, which expanded through membrane recapture after TRH stimulation. FM1-43 could therefore be a useful probe for membrane cycling associated with secretory responses.     

单纯疱疹病毒通用及Ⅱ型特异性DNA探针的制备及应用研究

单纯疱疹病毒（ＨＳＶ）Ⅰ型及Ⅱ型之间有很多共同抗原,能引起血清学交叉反应,鉴别诊断比较困难。本实验利用重组ＤＮＡ技术,将部分ＨＳＶ－２ＤＮＡ的ＰｓｔＩ片段克隆到载体质粒ＰＳＫ中,并筛选出两个重组质粒（Ｐ和Ｐ）只与ＨＳＶ－２反应,与ＨＳＶ－１不反应,这两个重组质粒中所含的ＨＳＶ－２ＤＮＡ片段大小分别是３．１和４．３ｋｂ,另外,还筛选了一个重组质粒（ＰＨＳＶ２－１,含５．８ｋｂＨＳＶ－２ＤＮＡ片段）与ＨＳＶ－１和ＨＳＶ－２均反应。将４．３ｋｂ的片段用光生物素标记后作为探针检测了１５９份人阴道拭子,其中２３份样品呈阳性反应,其余均为阴性,从２３份阳性样品中挑选１２价涂片用间接荧光抗体法检测也都呈阳性反应,随机挑选的几份杂交反应阴性样品在间接荧光试验中也是阴性。本实验制备的ＨＳＶ通用及ＨＳＶ－２型特异性探针将比常规的血清学方法诊断和鉴别ＨＳＶ－１和ＨＳＶ－２感染更为可靠。     

生物素标记HBV RNA探针的制备及应用

本文首次采用ＳＰ６５特殊质粒与人类的乙型肝炎病毒ＤＮＡ重组,制备了Ｂｉｏ－ＨＢＶ ＲＮＡ探针,能特异地与ＨＢＶ ＤＮＡ杂交,将该探针与缺口转移方法标记的Ｂｉｏ－ＨＢＶ ＤＮＡ探针进行了比较,结果显示出Ｂｉｏ－ＨＢＶ ＲＮＡ探针比Ｂｉｏ－ＨＢＶ ＤＮＡ探针的敏感性提高１０倍,并分别应用两种探针同时检测７０例乙肝病人血清中ＨＢＶ ＤＮＡ,阳性率各为３１．４２％、２８．５７％（Ｐ＞０．２５）。对Ｂｉｏ－     

Slow cortical potential biofeedback and the startle reflex

The negativity of slow cortical potentials (SCP) of the surface EEG is a measure of brain excitability, correlating with motor and cognitive preparation. Selfcontrol of SCP positivity has been shown to reduce seizure activity. Following SCP biofeedback from a central EEG electrode position, subjects gained bidirectional control over their SCP. The current study used a modified feedback methodology, and found a positive relationship between negativity and magnitude of EMG startle response (a measure of cortical and subcortical arousal, particularly aversive response disposition). Greater success in SCP differentiation was associated with self-report of less relaxation during negativity training.This research was supported by the Deutsche Forschungsgemeinschaft under grant No. SFB 307.     

Conjugates of adenosine mimetics and arginine-rich peptides serve as inhibitors and fluorescent probes but not as long-lifetime photoluminescent probes for protein arginine methyltransferases

The conjugates of an adenosine mimetic and oligo-l -arginine or oligo-d -arginine (ARCs) were initially designed in our research group as inhibitors and photoluminescent probes targeting basophilic protein kinases. Here, we explored a panel of ARCs and their fluorescent derivatives in biochemical assays with members of the protein arginine methyltransferase (PRMT) family, focusing specifically on PRMT1. In the binding/displacement assay with detection of fluorescence anisotropy, we found that ARCs and arginine-rich peptides could serve as high-affinity ligands for PRMT1, whereas the equilibrium dissociation constant values depended dramatically on the number of arginine residues within the compounds. The fluorescently labeled probe ARC-1081 was displaced from its complex with PRMT1 by both S-adenosyl-l -methionine (SAM) and S-adenosyl-l -homocysteine (SAH), indicating binding of the adenosine mimetic of ARCs to the SAM/SAH-binding site within PRMT1. The ARCs that had previously shown microsecond-lifetime photoluminescence in complex with protein kinases did not feature such property in complex with PRMT1, demonstrating the selectivity of the time-resolved readout format. When tested against a panel of PRMT family members in single-dose inhibition experiments, a micromolar concentration of ARC-902 was required for the inhibition of PRMT1 and PRMT7. Overall, our results suggest that the compounds containing multiple arginine residues (including the well-known cell-penetrating peptides) are likely to inhibit PRMT and thus interfere with the epigenetic modification status in complex biological systems, which should be taken into consideration during interpretation of the experimental data.     

Recent advances in the development of peptide-based inhibitors targeting epigenetic readers of histone lysine acetylation and methylation marks

Inhibitors for epigenetic readers of histone modifications are useful chemical probes to interrogate the functional roles played by their cognate targets in epigenetic regulation and can even serve as drugs for the treatment of diseases associated with the dysregulated targets. However, many epigenetic readers are intractable to small molecules, as the recognition of modified histone peptides commonly involves flat and extended protein surfaces. In contrast, the relatively large sizes and structural complexity of peptides help them achieve tight and specific binding to the target proteins. Increasing efforts have been made to target epigenetic readers using peptide-based inhibitors that can complement small molecules. In this review, we discuss the recent advances in the development of peptide-based inhibitors of lysine acetylation and methylation readers.     

Detection of Naegleria fowleri cysts in environmental samples by using a DNA probe

Abstract In this study we tried to detect DNA Naegleria fowleri in artificially contaminated environmental samples, with or without sediments, containing 10⁴ cysts of this pathogenic amoeba. We used two assays to extract DNA from samples: first, direct DNA extraction, which gave positive results only for water samples without sediment; second, DNA extraction after sample incubation on agar plates, which allowed us to remove amoeba growing out of the sediments, and which gave positive results for all samples, even those initially with sediments (5, 500 or 500 mg). Thus, this molecular identification appears as a powerful tool to investigate N. fowleri growth in environmental samples.     

A PCR primer-specific to Cylindrocarpon heteronema for detection of the pathogen in apple wood

Abstract An oligonucleotide primer (ChInt) was synthesised from the variable internally transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) of Cylindrocarpon heteronema . PCR with primers ChInt and ITS4 (from a conserved sequence of the rDNA) amplified a 470-bp fragment from several isolates of C. heteronema but not from various apple wood saprophytes. Amplification of this fragment was achieved from 1–2 pg of fungal DNA. These primers amplified a fragment of the same size from DNA extracted from cankered wood but only after impurities were removed from the DNA on a Qiagen tip-5 column. Southern hybridization analysis confirmed the 470-bp fragment from C. heteronema DNA and cankered wood to be identical.     

Mitochondrial DNA as a genetic marker for brown trout, Salmo trutta L., populations

Mitochondrial DNA was examined in natural and hatchery-reared stocks of brown trout, using different methods of restriction analysis. The methods included the development of a brown trout mt DNA hybridization probe through cloning part of the brown trout mitochondrial genome. In addition, fragments were analysed by ethidium bromide staining and end-labelling. The relative merits of each of these methods in assessing levels of genetic relatedness between the natural and hatchery-reared brown trout stocks were evaluated. In addition, the study revealed a diagnostic mtDNA restriction pattern which could be used as a genetic marker for the discrimination of these two groups of brown trout.     

The prevalence of Fasciola hepatica in its snail intermediate host determined by DNA probe assay

Accurate snail intermediate host infection prevalence data have the potential to be extremely useful in determining seasonal transmission dynamics of Fasciola hepatica. Because the microscopic techniques currently used lack the sensitivity and specificity necessary to obtain meaningful infection prevalence data, we developed a highly accurate and efficient DNA probe assay. The assay has a sensitivity of 100%, a specificity of >99%, easily detects a single miracidia and does not cross-hybridize with DNA of Fascioloides magna, Paramphistomum liorchis or Heterobilharzia americana, trematodes that share the same intermediate host and enzootic range as Fasciola hepatica. Using this assay, we determined the prevalence of F. hepatica in its snail intermediate host, Fossaria cubensis, during the second year of a 2-year study on the epizootiology of Fasciola hepatica in Florida. The overall infection prevalence of snails assayed in this study (n = 5246) was 1.5% and ranged from 0.1% to 3.1% for individual cattle ranches. Additionally, infection prevalence differed significantly for successive size groupings of snails, varying from 0% for 1-mm snails to 18.5% for 9- and 10-mm snails. The accuracy and efficiency of the DNA probe assay reported here for determining snail infection prevalence offers an inexpensive alternative to tracer animal studies for determining the epizootiology of F. hepatica.