Direct in situ reverse transcriptase polymerase chain reaction for detection of measles virus

Behavioral contexts can evoke a variety of autonomic modes of response, characterized by reciprocal, coactive, or independent changes in the autonomic divisions. The present study investigated the modes of autonomic response to visual illusion and mental arithmetic tasks, by the use of noninvasive measures of sympathetic (pre-ejection period; PEP) and parasympathetic (respiratory sinus arrhythmia; RSA) cardiac control. As previously demonstrated, mental arithmetic was associated with a reciprocal pattern of sympathetic activation and vagal withdrawal. The illusion task, however, yielded a distinct mode of vagal activation in the absence of sympathetic change. Responses within tasks were reliable. In contrast to the general intertask consistency reported for stress tasks that yield similar autonomic modes of response, however, neither PEP nor RSA responses were correlated across the illusion and arithmetic tasks. This may be attributable to the dissimilar modes of autonomic control evoked by these tasks. The distinct modes of autonomic response to arithmetic and illusions emphasize the importance of a bivariate model of autonomic control, and may offer important experimental tools for psychophysiological studies of autonomic control.     

A reverse transcriptase polymerase chain reaction assay for the detection of thermophilic Campylobacter spp

The purpose of this study was to determine the most appropriate method of normalisation for dual fine wire electromyography of shoulder muscles. Five healthy subjects were studied, with one muscle investigated in each subject (2 supraspinatus, 2 infraspinatus, 1 subscapularis). Three dual fine wire electrodes were inserted 1 cm apart around the recognised insertion points. Each subject performed five types of cyclic exercise on an isokinetic muscle dynamometer with an isometric maximal voluntary contraction (MVC) being performed before and after the exercise protocol. The EMG signal was normalised using each of the MVC voltage, the peak voltage and the whole-cycle mean voltage. There was a considerable difference (5-143%) between the MVC signals pre- and post-protocol, although no systematic trend was demonstrable. The overall mean between electrode variation in the normalised signal measured at the peak of the cycle ranged from 48-71% when normalised to pre-protocol MVC, but only 4-13% when normalised to the peak voltage and 9-17% using the whole-cycle mean voltage. However the pattern of activation within the movement cycle, which was preserved by normalisation using the peak or mean signal, was consistent between different electrode positions. It was concluded that the EMG signal depended on electrode position even when near the recognised insertion point, and that the MVC signal is highly variable in magnitude between electrodes and between pre- and post-protocol measurements.     

Diagnosis of Prader-Willi syndrome by reverse transcriptase polymerase chain reaction

To approve Prader-Willi syndrome by molecular diagnostic assay, polymerase chain reaction of reverse-transcribed RNA was introduced by which indirect information can be gained on all known forms of the mutation. In this pilot study, 4 patients and 16 healthy control individuals were examined. Although the different mutation forms can not directly by identified by this approach, it is a useful and reliable test to confirm the clinical diagnosis of the Prader-Willi syndrome, and to screen for the syndrome in patients who present with only a few typical features.     

RNA extraction from virus-diseased sweet potato for reverse transcriptase polymerase chain reaction analysis

Apoptosis occurs in both clinical and experimental alcoholic liver disease. The mechanisms involved in alcohol-induced apoptosis of liver cells are not completely understood. Induction of cytochrome P450 2E1, the alcohol-inducible cytochrome P450, is one of the proposed mechanisms. Exposure of Hep G2 cells expressing cytochrome P450 2E1 to arachidonic acid leads to increased lipid peroxidation and apoptosis. Increased levels of iron in the liver also promote lipid peroxidation and are associated with increased numbers of apoptotic hepatocytes. Tumor necrosis factor (TNF) acting through its receptors can induce apoptosis in hepatocytes. Increased levels of tumor necrosis factor and its receptors have been described in alcoholic liver disease. The liver is also CD95 receptor positive and in liver tissue from patients with alcoholic hepatitis, the CD95 ligand is expressed at high levels in hepatocytes. Cytotoxic T lymphocytes could, through the CD95 receptor-ligand interaction, promote apoptosis.     

Rapid detection of thymidylate synthase gene expression levels by semi-quantitative competitive reverse transcriptase polymerase chain reaction followed by quantitative digital image analysis

We describe a simplified and reliable polymerase chain reaction (PCR) method to quantify thymidylate synthase (TS) gene expression levels from clinical human tumor biopsy samples as small as 100 mg using the beta-actin housekeeping gene as a reference standard. The semiquantitative RT-PCR is carried out by the coamplification of the target template and an external competitor using primer pairs common to both templates in the same reaction vessel. Quantitative digital image analysis is performed directly after electrophoresis, thus mRNA quantification is done quickly and without the use of radioactive substances. The observed relative TS gene expression levels varied between 3- and 40-fold, but most of the values were grouped within a 10-fold range. There is an observed 89% correlation between TS mRNA expression and protein levels. These findings suggest that preliminary experiments used to determine the linear range of RT-PCR amplification in non-competitive semiquantitative PCR experiments, and the use of radioactive substances to quantify PCR products may be unnecessary.     

Highly sensitive and specific polymerase chain reaction assays for detection of baboon and pig cells following xenotransplantation in humans

Pig and baboon xenotransplants in humans require assays that discriminate source from human cells to investigate engraftment and identify true infection of recipients with xenogeneic endogenous retroviruses. We developed two polymerase chain reaction assays that target a porcine-specific sequence in the beta-globin gene and a baboon-specific sequence in the mitochondrial cytochrome oxidase subunit II gene. The sensitivity and specificity of both assays were evaluated on DNA lysates from baboon, pig, and human peripheral blood lymphocytes. Both assays detected single cells in backgrounds of human DNA. Additionally, both assays were highly specific and yielded negative results in reactions containing only human DNA. The two assays reliably detected peripheral blood lymphocyte samples from 14 baboons and 16 pigs. The baboon- and pig-specific target sequences are gender independent and nonpolymorphic and allow universal applicability. The high sensitivity and specificity is of particular importance in assessing low-level engraftment in human xenotransplant chimeras.     

Detection of contamination of vaccines with the reticuloendotheliosis virus by reverse transcriptase polymerase chain reaction (RT-PCR)

Phospholipase C (phosphatidylcholine phosphohydrolase, EC 3.1.4.3) and lipase (EC 3.1.1.3) activities were detected in the supernatant fluid of Pseudomonas fluorescens strain D cultures. A combination of ultrafiltration and successive chromatography through columns of Sephadex G-75 and DEAE-cellulose was used to purify the phospholipase C over 700-fold from the culture medium, with 28.5% yield. The purified enzyme appeared as a single band after polyacrylamide gel electrophoresis. The apparent molecular mass of the phospholipase C was 36,000 daltons when estimated by gel permeation chromatography. The purified enzyme hydrolysed phosphatidylcholine more efficiently than phosphatidylethanolamine. The synthetic substrate p-nitrophenylphosphorylcholine, phosphatidylinositol or sphingomyelin were not hydrolysed. Hydrolysis of phosphatidylcholine was inhibited by EDTA (1mM) and stimulated by Zn2+, Mg2+ ions and detergents. These properties of the enzyme indicate that it is distinct from the previously reported Ps. fluorescens phospholipase C.     

Specificity of the BAX polymerase chain reaction system for detection of the foodborne pathogen Listeria monocytogenes

PURPOSE: The purpose of this study was to evaluate the toxicity and efficacy of twice-daily external irradiation to the pelvis and para-aortics with brachytherapy and concurrent chemotherapy for carcinoma of the cervix with positive para-aortic lymph nodes. METHODS AND MATERIALS: This study was designed to administer twice-daily radiation doses of 1.2 Gy to the pelvis and para-aortics at 4- to 6-h intervals, 5 days per week. The total external radiation doses were 24 to 48 Gy to the whole pelvis, 12 to 36 Gy parametrial boost, and 48 Gy to the para-aortics with an additional boost to a total dose of 54 to 58 Gy to the known metastatic para-aortic site. One or two intracavitary applications were performed to deliver a total minimum dose of 85 Gy to point A. Cisplatin (75 mg/m2, days 1 and 22) and 5-FU (1000 mg/m2/24 h x 4 days; days 1 and 22) were given for two or three cycles. RESULTS: Twenty-nine patients with clinical Stages I to IV carcinoma of the cervix with biopsy-proven para-aortic lymph nodes were enrolled in this study. Hyperfractionated external radiotherapy was completed in 86% (25 of 29). Brachytherapy was given in two applications to 48% (14 of 29), 31% (9 of 29) had one intracavitary application, 14% (4 of 29) had no brachytherapy, one had three applications, and one had five HDR applications. Radiotherapy was completed per protocol in 69%. Three courses of chemotherapy were given to 24% (7 of 29), 72% (21 of 29) received two courses, and one patient did not receive chemotherapy. The acute toxicity from chemotherapy was Grade 1 in 3%, Grade 2 in 17%, Grade 3 in 48%, and Grade 4 in 28%. Radiotherapy toxicity was Grade 1 in 7%, Grade 2 in 34%, Grade 3 in 21%, and Grade 4 in 28%. One Grade 5 toxicity occurred and the patient died from a myocardial infarction from chemotherapy and radiotherapy colitis during her course of therapy. The median follow-up time was 18.9 months. The overall survival estimates were 59% at 1 year and 47% at 2 years. The probability of local-regional failure was 38% at 1 year and 49% at 2 years. The probability of disease failure at any site was 45% at 1 year and 59% at 2 years. CONCLUSION: The results suggest that twice-daily external irradiation to the pelvis and para-aortics with brachytherapy and concurrent chemotherapy resulted in an unacceptably high rate (31%, 9 of 29) of Grade 4 nonhematologic toxicity. One patient died from complications of therapy. Radiotherapy was completed per protocol in 69%. The survival estimates appear no better than standard fractionation radiotherapy without chemotherapy. Additional follow-up is necessary for long-term survival estimates.     

Detection of a mutator phenotype in cancer cells by inter-Alu polymerase chain reaction

A mutator phenotype due to a DNA mismatch repair deficiency is usually detected by typing a number of microsatellite markets. Here, eight hereditary nonpolyposis colon cancer patients with microsatellite instability were investigated by inter-Alu PCR, known to amplify DNA segments that may represent preferential targets of replication errors. Among 40-60 bands revealed in a single PCR experiment, more than 20% were found altered in tumoral DNA samples compared to matched normal samples from the same patient. Shifts and changes in signal intensity accounted for most of the alterations, whereas gains or losses of bands were rare. Certain bands were affected only in a single patient, whereas the instabilities in others were common. These results suggest that some genomic regions are more susceptible than others to the expression of a mutator phenotype. Four such bands altered in at least five patients were characterized further and shown to be unstable because of contractions of the Alu poly(A) tails. Interestingly, none of the bands representing loci shown previously to be polymorphic in the population displayed instability in the tumoral samples. Inter-Alu PCR appears to be a robust, cost-effective, and sensitive technique for revealing the mutator phenotype in cancer cells.     

Detection of circulating tumor cells in patients with prostate cancer using prostate specific membrane-derived primers in the polymerase chain reaction

Angiotensin-converting enzyme (ACE) is known to play an important role in the pathobiology of human essential hypertension. Similarly, cis-unsaturated fatty acids, prostaglandins, and free radicals are believed to play a role in the control of blood pressure. It was observed that all the cis-unsaturated fatty acids tested can inhibit ACE activity to a significant degree. On the other hand, prostaglandins and free radicals, superoxide anion, hydrogen peroxide, and hydroxyl radical did not show significant inhibitory effects on ACE activity in vitro. But, the nitric oxide donor sodium nitroprusside showed a potent inhibitory action on ACE activity, suggesting that one of the possible mechanism(s) by which nitric oxide can bring about its anti-hypertensive action might be by modulating ACE activity in addition to its direct vasodilator action. These results indicate that there is a close interaction among ACE activity, cis-unsaturated fatty acids, and nitric oxide, which may have relevance to the pathobiology of human essential hypertension.