Use of the Roche LightCycler instrument in a real-time PCR for Trichomonas vaginalis in urine samples from females and males

Trichomonas vaginalis is the agent of a highly prevalent sexually transmitted infection (STI) that can result in vaginitis, urethritis, and preterm birth. Traditional methods of diagnosis, including wet preparation, can be unreliable. In this study, we describe the adaptation of an existing PCR method for specific detection of T. vaginalis DNA into a rapid real-time PCR assay based on fluorescence resonance energy transfer (FRET) probe chemistry. The FRET-based assay described demonstrated high sensitivity with a detection limit of 1.06 organisms, as well as a high specificity. A total of 253 urine samples collected prospectively from both men and women were tested for T. vaginalis DNA with both the FRET-based assay and a previously validated PCR assay. When the validated PCR assay was used as the "gold standard" and after discrepancies had been resolved, our FRET-based assay demonstrated an analytical sensitivity and specificity of 90.1 and 100%, respectively. Overall results suggest that FRET-based assays can provide rapid, accurate, and high-throughput detection of T. vaginalis and may prove useful in clinical settings and for large-scale screening programs.     

A computer-interfaced photometer and systematic spacing of duplicates to control within-plate enzyme-immunoassay variation

A Multiskan photometer for reading microtiter plate enzyme immunoassays was linked with a time sharing computer to facilitate control of assay variation and analysis of results. The interface that converted photometer output to RS-232-C format required changes to divide the output into segments short enough for input to the computer. To measure within-plate variation and investigate how the method of allocating sample duplicates to plate wells may affect the estimation of sample variance, uniformity tests were conducted with 47 plates. Coefficients of variation (CV) among wells within-plates ranged from 4.6 to 20.7% and in two-thirds of the plates exceeded 10%. Duplicates allocated to adjacent wells (method 1) gave consistently higher CV for sample means than duplicates allocated to opposite plate quadrants (method 2). In general, the CV by method 2 was about 30% smaller than that by method 1. Analysis of variance confirmed the effectiveness of the quadrant pattern of duplicate allocation as a method of controlling variation that arises from well position effects.     

Characterization of virus-like particles assembled in a recombinant baculovirus system expressing the capsid protein of a fish nodavirus

Infectious bursal disease virus (IBDV) causes severe immunodeficiency in young chickens by destroying the precursors of antibody-producing B cells in the bursa of Fabricius. It has been shown that IBDV infection induces apoptosis in chicken embryo and tissue culture cells. We previously reported that an IBDV mutant lacking the expression of 17-kDa nonstructural (NS) protein exhibited decreased apoptotic effects in cell culture as compared to the parental IBDV, suggesting that the NS protein may be involved in induction of apoptosis. Here, we report that the NS protein of IBDV alone is capable of inducing apoptosis in cell culture. Transfection of chicken B-lymphocyte cell line (RP9) and chicken embryo fibroblast cells with a plasmid DNA, containing the NS protein gene under the control of the immediate-early promoter-enhancer region of human cytomegalovirus, induced programmed cell death in both cell lines. Apoptosis changes, such as chromatin condensation, DNA fragmentation, and the appearance of apoptotic nuclear bodies, were observed in cell cultures 48-h posttransfection. As reported earlier, the mutant IBDV grew to lower titers with slower replication kinetics and lower cytopathogenicity when compared to that of the parental virus. Here, we demonstrate that the mutant virus is closely associated with cells and its yield from the supernatant was approximately 30-fold lower than the wild-type due to increased cell association, indicating a deficiency in lysis of virus-infected cells. Taken together, our results indicate that the NS protein of IBDV is highly cytotoxic, which brings about the release of the viral progeny from cells, and thus play an important role in viral pathogenesis.     

Simultaneous assay of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human plasma using normal-phase liquid chromatography-tandem mass spectrometry with a silica column and an aqueous organic mobile phase

Morphine (MOR) is an opioid analgesic used for the treatment of moderate to severe pain. MOR is extensively metabolized to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). A rapid and sensitive method that was able to reliably detect at least 0.5 ng/ml of MOR and 1.0 ng/ml of M6G was required to define their pharmacokinetic profiles. An LC-MS-MS method was developed in our laboratory to quantify all three analytes with the required sensitivity and a rapid turnaround time. A solid-phase extraction (SPE) was used to isolate MOR, M3G, M6G, and their corresponding deuterated internal standards from heparinized plasma. The extract was injected on a LC tandem mass spectrometer with a turbo ion-spray interface. Baseline chromatographic separation among MOR, M3G, and M6G peaks was achieved on a silica column with an aqueous organic mobile phase consisting of formic acid, water, and acetonitrile. The total chromatographic run time was 3 min per injection, with retention times of 1.5, 1.9 and 2.4 min for MOR, M6G, and M3G, respectively. Chromatographic separation of M3G and M6G from MOR was paramount in establishing the LC-MS-MS method selectivity because of fragmentation of M3G and M6G to MOR at the LC-MS interface. The standard curve range in plasma was 0.5-50 ng/ml for MOR, 1.0-100 ng/ml for M6G, and 10-1000 ng/ml for M3G. The inter-day precision and accuracy of the quality control (QC) samples were <7% relative standard deviation (RSD) and <6% relative error (R.E.) for MOR, <9% RSD and <5% R.E. for M6G, and <3% RSD and <6% R.E. for M3G. Analyte stability during sample processing and storage were established. Method ruggedness was demonstrated by the reproducible performance from multiple analysts using several LC-MS-MS systems to analyze over one thousand samples from clinical trials.     

Age-dependent and iron-independent expression of two mRNA isoforms of divalent metal transporter 1 in rat brain

The DMT1(Nramp2/DCT1) is a newly discovered proton-coupled metal-ion transport protein. The cellular localization and functional characterization of DMT1 suggest that it might play a role in physiological iron transport in the brain. In the study, we evaluated effects of dietary iron and age on iron content and DMT1 expression in four brain regions: cortex, hippocampus, striatum, substantia nigra. Total iron content in all regions was significantly lower in the low-iron diet rats and higher in the high-iron diet rats than that in the control animals, showing that dietary iron treatment for 6-weeks can alter brain iron levels. Contrary to our expectation, there was no significant alternation in DMT1(+IRE) and (-IRE) mRNA expression and protein content in all brain regions examined in spite of the existence of the altered iron levels in these regions after 6-weeks' diet treatment although TfR mRNA expression and protein level were affected significantly, as was expected. The data demonstrates that expression of DMT1(+IRE) and (-IRE) was not regulated by iron in these regions of adult rats. The lack of response of DMT1 to iron status in the brain suggests that the IRE of brain DMT1 mRNA might be not really iron-responsive and that DMT1-mediated iron transport might be not the rate-limiting step in brain iron uptake in adult rats. Our findings also showed that development can significantly affect brain iron and DMT1(+IRE) and (-IRE) expression but the effect varies in different brain regions, indicating a regionally specific regulation in the brain.     

Absence of a prominent Th2 cytokine response in human tuberculosis.

Depressed Th1 responses are a prominent feature of human tuberculosis, but an enhanced Th2 response has not been detected in peripheral blood T cells stimulated in vitro with Mycobacterium tuberculosis. In disease due to Mycobacterium leprae, Th2 cells predominate in tissue lesions of patients with extensive disease but are absent from peripheral blood. To determine if Th2 cells are present in tissue lesions of tuberculosis patients, we evaluated patterns of cytokine expression in lymph nodes from tuberculosis patients with or without human immunodeficiency virus infection and in controls without tuberculosis. Gamma interferon and interleukin-10 (IL-10) mRNA expression in tuberculosis patients with or without human immunodeficiency virus infection was high, whereas IL-4 expression in the same patients was low. Immunolabeling studies showed that macrophage production of IL-12 was increased in lymph nodes from tuberculosis patients, that gamma interferon was produced by T cells, and that IL-10 was produced by macrophages rather than Th2 cells. These results indicate that Th2 responses are not enhanced either systemically or at the site of disease in human tuberculosis.     

脑缺血再灌流对大鼠海马FOS蛋白的诱导及电针对其的影响

目的　探讨脑缺血再灌流后电针对大鼠海马FOS蛋白表达的影响。方法　采用夹闭大鼠双侧颈总动脉造成的脑缺血再灌流损伤模型 ,电针“百会”、“风池”、“大钟”及“足三里”穴 ,频率 2～ 2 0Hz,强度以肌肉轻微抖动为准 ,持续 30min ,4h后观察海马FOS蛋白的表达。结果　电针能明显加强脑缺血再灌流后海马各区FOS蛋白的表达。结论　缺血再灌流可诱导海马FOS蛋白的显著表达 ;电针信息对缺血后海马神经元的功能可能会有影响。     

Poly(D,L-lactic acid) surfaces modified by silk fibroin: effects on the culture of osteoblast in vitro.

The objective of this study was to modify the surface of poly(D,L-lactic acid) (PDLLA) with different molecular weight of silk fibroins, and assess the effects of the modified surfaces on the functions of rat osteoblasts cultured in vitro. The properties of the modified PDLLA surface and the control one were investigated by contact angle and electron spectroscopy for chemical analysis (ESCA). The former indicated the variation of hydrophilicity and the latter suggested that the modified PDLLA film using silk fibroin is enriched with nitrogen atoms. The biocompatibility of the PDLLA film may be altered and in turn affects the seeded cell functions. Therefore, attachment and proliferation of osteoblasts seeded on the modified PDLLA films and the control one were examined. Cell morphologies on these films were studied by scanning electron microscopy (SEM) and cell viability was evaluated by MTT assay. In addition, differentiated cell function was assessed by measuring the alkaline phosphatase (ALP) activity. These results suggest that the silk fibroin-modified PDLLA surface can improve the interaction between osteoblasts and the PDLLA films.     

Localization of the novel Xin protein to the adherens junction complex in cardiac and skeletal muscle during development.

Previously, we demonstrated that chick embryos treated with antisense oligonucleotides against a striated muscle-specific Xin exhibit abnormal cardiac morphogenesis (Wang et al. [1999] Development 126:1281-1294); therefore, we surmised a role for Xin in cardiac development. Herein, we examine the developmental expression of Xin through immunofluorescent staining of whole-mount mouse embryos and frozen heart sections. Xin expression is first observed within the heart tube of embryonic day 8.0 (E8.0) mice, exhibiting a peripheral localization within the cardiomyocytes. Colocalization of Xin with both beta-catenin and N-cadherin is observed throughout embryogenesis and into adulthood. Additionally, Xin is found associated with beta-catenin within the N-cadherin complex in embryonic chick hearts by coimmunoprecipitation. Xin is detected earlier than vinculin in the developing heart and colocalizes with vinculin at the intercalated disc but not at the sarcolemma within embryonic and postnatal hearts. At E10.0, Xin is also detected in the developing somites and later in the myotendon junction of skeletal muscle but not within the costameric regions of muscle. In cultured C2C12 myotubes, the Xin protein is found in many speckled and filamentous structures, coincident with tropomyosin in the stress fibers. Additionally, Xin is enriched in the regions of cell-cell contacts. These data demonstrate that Xin is one of the components at the adherens junction of cardiac muscle, and its counterpart in skeletal muscle, the myotendon junction. Furthermore, temporal and spatial expressions of Xin in relation to intercalated disc proteins and thin filament proteins suggest roles for Xin in the formation of cell-cell contacts and possibly in myofibrillogenesis.     

Distinct protein degradation mechanisms mediated by Cul1 and Cul3 controlling Ci stability in Drosophila eye development

The ubiquitin-like protein, Nedd8, covalently modifies members of the Cullin family. Cullins are the major components of a series of ubiquitin ligases that control the degradation of a broad range of proteins. We found that Nedd8 modifies Cul1 in Drosophila. In Drosophila Nedd8 and Cul1 mutants, protein levels of the signal transduction effectors, Cubitus interruptus (Ci) and Armadillo (Arm), and the cell cycle regulator, Cyclin E (CycE), are highly accumulated, suggesting that the Cul1-based SCF complex requires Nedd8 modification for the degradation processes of Ci, Arm, and CycE in vivo. We further show that two distinct degradation mechanisms modulating Ci stability in the developing eye disc are separated by the morphogenetic furrow (MF) in which retinal differentiation is initiated. In cells anterior to the MF, Ci proteolytic processing promoted by PKA requires the activity of the Nedd8-modified Cul1-based SCF(Slimb) complex. In posterior cells, Ci degradation is controlled by a mechanism that requires the activity of Cul3, another member of the Cullin family. This posterior Ci degradation mechanism, which partially requires Nedd8 modification, is activated by Hedgehog (Hh) signaling and is PKA-independent.