In vitro amplification of human basic fibroblast growth factor mRNA by RNA-specific exon-junction primers

We hypothesized that left ventricular (LV) cavity size measured on dipyridamole thallium scintigraphy identifies patients at risk for late nonfatal myocardial infarction and cardiovascular death. Accordingly, we retrospectively evaluated the predictive value of clinical and scintigraphic variables, including transendocardial LV cavity measurement performed on formatted images, in 335 vascular surgery patients. A nonhomogeneous perfusion pattern and enlarged LV cavity size were the most significant predictors of late events, and the interaction between these two variables was more predictive than was either variable alone. Life-table analysis demonstrated that patients with normal perfusion patterns had the lowest incidence of late events regardless of cavity size (p < 0.0005). Conversely, patients with a nonhomogeneous perfusion pattern and the largest LV cavity measurements were at the highest risk for late cardiac events (p < 0.0001). Therefore, this study demonstrated that a measurement of LV scintigraphic cavity size can provide important risk stratification for late cardiac events.     

In vitro evolution of molecular cooperation in CATCH, a cooperatively coupled amplification system

BACKGROUND: One of the key issues in the investigation of evolution is how complex systems evolved from simple chemical replicators. Theoretical work proposed several models in which complex replicating systems are kinetically stabilized. The development of powerful isothermal amplification technique allows complex nucleic acid based evolving in vitro systems to be set up, which may then serve to verify experimentally current theories of evolution. Recently such a system based on the 3SR (self-sustained sequence replication) reaction has been established to investigate the evolution of cooperation: the trans-cooperatively coupled CATCH (cooperative amplification by cross hybridization). RESULTS: Over four rounds of serial transfer, the cooperatively coupled two species CATCH system evolved into a more complex cooperative four species system, which then was overgrown by CATCH-derived RNA-Z-like hairpin species. In contrast to the classical RNA-Z species, these molecules have complementary loop sequences and self-amplify using a dual mechanism that includes concentration-dependent phases of noncooperative and cooperative amplification. CONCLUSIONS: The evolution of a cooperative system, under conditions that were alternately unfavorable and favorable for cooperative amplification, led to a system showing facultative cooperation. This principle of facultative cooperation preserves the complexity of the system investigated and could have general implications for the evolution and stabilization of cooperation under oscillating reaction conditions.     

Ex vivo clonotype primer-directed gene amplification to identify malignant T cell repertoires

The effect of weight on mortality was examined using data from the first National Health and Nutrition Examination Survey (NHANES I) Epidemiologic Follow-up Study for white women aged 65 to 74 years at baseline. There was a U-shaped curve relating the Quetelet index categories to total mortality, with increased risk for both lean and heavy women. However, the increased risk to lean subjects occurred only among those who had lost more than 8.55% from their reported lifetime maximum weight. Controlling for baseline medical conditions, excluding early years of follow-up, and limiting the analysis to never-smokers did not greatly change the results. Lean women with stable weight have the lowest risk of mortality, while those who have lost weight have a high risk. Heavy women have a high risk of mortality regardless of weight-loss history. Thus, the effect of weight on mortality is modified by history of weight loss in older women, even when accounting for factors associated with weight loss and increased mortality risk.     

Establishment and characterization of a megakaryoblast cell line with amplification of MLL

A new cell line with megakaryoblastic features, designated UoC-M1, was established from the malignant cells of a 68-year-old patient with acute myeloid leukemia. The patient's leukemic cells reacted with alpha-naphthyl acetate esterase and acid phosphatase and expressed CD7, CD24, CD34, CD38, CD45, HLA-DR and CD61. Cytogenetic analysis of the patient's malignant cells (and of the UoC-M1 cells) showed a human, male hypodiploid karyotype with many chromosome rearrangements and marker chromosomes. Spectral karyotyping (SKY) analysis complemented the G-banded karyotyping and clarified several chromosomal translocations and identified the marker chromosomes. Fluorescence in situ hybridization (FISH) and SKY analysis demonstrated that one marker chromosome contained three segments of chromosome 9 interspersed with three segments of chromosome 11, as well as a portion of chromosome 19. FISH analysis with a probe for MLL revealed that the UoC-M1 cells contained four copies of the MLL gene. Southern blot analysis determined that the MLL gene had a germline profile while Northern and Western analyses showed that the MLL mRNAs and protein were of the appropriate sizes. This is the first report of amplification of the MLL gene which may be an additional mechanism of leukemogenesis or disease progression.     

DNA amplification

The polymerase chain reaction has become a mainstream tool for the molecular biologist. The sensitivity, efficiency, and speed of this method is unparalleled for the amplification and detection of exquisitely minute quantities of nucleic acids. Through repetitive cycles of heat denaturation of samples, followed by the base pairing of primers designed to identify one DNA sequence among the cellular heterogeneity, and finally synthesis of new DNA strands identical to the target, single molecules and individual genes can be detected and subsequently characterized. This method has revolutionized the study of gene organization, structure, and expression, not to mention offering newer, faster, and more economical means for the clinical detection infectious disease. That PCR has been fruitful is undisputed; however, the method is not without shortcomings. Among the major limitations of this method are the absolute requirement for well-designed primers, the super sensitivity of this method to biological contaminants from any of a variety of sources, and subtle, though very important, inter- and intra-laboratory variations in technique.     

Gene amplification in a p53-deficient cell line requires cell cycle progression under conditions that generate DNA breakage

Amplification of genes involved in signal transduction and cell cycle control occurs in a significant fraction of human cancers. Loss of p53 function has been proposed to enable cells with gene amplification to arise spontaneously during growth in vitro. However, this conclusion derives from studies employing the UMP synthesis inhibitor N-phosphonacetyl-L-aspartate (PALA), which, in addition to selecting for cells containing extra copies of the CAD locus, enables p53-deficient cells to enter S phase and acquire the DNA breaks that initiate the amplification process. Thus, it has not been possible to determine if gene amplification occurs spontaneously or results from the inductive effects of the selective agent. The studies reported here assess whether p53 deficiency leads to spontaneous genetic instability by comparing cell cycle responses and amplification frequencies of the human fibrosarcoma cell line HT1080 when treated with PALA or with methotrexate, an antifolate that, under the conditions used, should not generate DNA breaks. p53-deficient HT1080 cells generated PALA-resistant variants containing amplified CAD genes at a frequency of >10(-5). By contrast, methotrexate selection did not result in resistant cells at a detectable frequency (<10(-9)). However, growth of HT1080 cells under conditions that induced DNA breakage prior to selection generated methotrexate-resistant clones containing amplified dihydrofolate reductase sequences at a high frequency. These data demonstrate that, under standard growth conditions, p53 loss is not sufficient to enable cells to produce the DNA breaks that initiate amplification. We propose that p53-deficient cells must proceed through S phase under conditions that induce DNA breakage for genetic instability to occur.     

In vitro generation of human dendritic cells and cell therapy

HHV-7 growth on Sup-T1, an immature T-cell line, was studied using different HHV-7 isolates obtained in our laboratory. Titration of viral yields showed that all the virus isolates propagate on this cell line more efficiently than in cord blood lymphocytes, the cells usually recommended for HHV-7 growth. The permissivity of Sup-T1 to HHV-6, whose ability to replicate in these cells was still unknown, was also investigated using two virus isolates representative of variants A and B respectively. Both isolates were able to propagate on Sup-T1 and viral titres were similar to those obtained in cord blood lymphocytes. As the efficient propagation of both HHV-7 and HHV-6 isolates in Sup-T1 cultures, these cells may replace more time consuming and expensive cord blood lymphocyte preparations for the propagation of both the viruses.     

In vivo detection of fluorescent tumor-specific cytotoxic T cell clones

A procedure for detecting mumps virus in under 48 h was developed using the PCR. The sensitivity of the PCR amplification reaction and of the detection of the PCR product was significantly improved by: (i) enriching for viral template RNAs by overnight culture of the virus in Vero cells and (ii) substitution of polyacrylamide gel analysis for agarose gel electrophoresis. The technique was capable of detecting 1-20 infectious units of virus or an equivalent of 1-10 pg of mumps virus-specific plasmid DNA.     

In vitro amplification of the 16S rRNA genes from Mycoplasma bovirhinis, Mycoplasma alkalescens and Mycoplasma bovigenitalium by PCR

Polymerase chain reaction (PCR) systems were developed for detection of 3 pathogenic bovine mycoplasmas, Mycoplasma alkalescens (Mak), M. bovigenitalium (Mbg) and M. bovirhinis (Mbr). The primers were selected from the sequences of the 16S rRNA from each of the mycoplasmas. Neither the Mbg-PCR system nor the Mbr-PCR system showed cross-amplification among 24 bovine, caprine-ovine and human mycoplasma species (including Acholeplasma and Ureaplasma species) tested. The Mak-PCR system showed cross-amplification with M. canadens ATCC29418T (T = type strain). The sensitivity of each PCR system to detect the proper mycoplasma was 10(3) colony forming units (CFU) when a pure culture was tested, and 2 x 10(3) CFU when the mycoplasma culture was spiked into a calf nasal swab sample. The target mycoplasmas in a clinical nasal swab sample that could be detected by the direct plate method could also be detected by these PCR systems.     

Catecholamine detection using enzymatic amplification

Different amplification sensors based on the substrate recycling principle were investigated with respect to their applicability to catecholamine detection. In the bioelectrocatalytic approach, glassy carbon electrodes were modified by laccase or a PQQ-dependent glucose dehydrogenase. Substrate recycling occurs and the detection limit is in the lower nanomolar concentration range (e.g. 10 nM dopamine and 1 nM noradrenaline for the laccase- and glucose dehydrogenase-modified electrodes, respectively). Combinations of glucose dehydrogenase with laccase or tyrosinase were investigated as bienzymatic probes. Among the systems we studied, the laccase/glucose dehydrogenase sensor is the most sensitive (detection limit: 0.5 nM adrenaline). The selectivities of the different sensor systems are discussed. Application of the laccase/glucose dehydrogenase electrode in different media (i.e. brain homogenate, heart effluate) was successfully shown. For samples with high concentrations of interfering substances (uric and ascorbic acid), the interferences can be effectively removed using enzymatic methods.     

Gene amplification in bladder tumors

Eight patients with transitional cell bladder carcinoma were analyzed viewing the detection of ras oncogene mutations. This sample was picked out from a preliminary study of 20 cases of transitional cell tumors where complex chromosomal rearrangements were observed in tumors of grade II/III. In this first stage of the study non invasive tumors presented an almost diploid chromosome number while the most infiltrative ones displayed pseudo triploidy, triploidy or tetraploidy. A Subsequent study searched for the mutational frequency of the oncogene n-ras using the knowledge of direct DN sequencing and based on the technology of polymerase chain reaction amplification (PCR) in view to establish a relationship between the mutational frequency and the tumor stage and grade. The wild type allele was present in all the tumors examined. Point mutations were detected in 1/3 of the analyzed samples. These samples displayed a grade II-III histology as opposed to the other tumors. These data appear to indicate that n-ras mutations may be early phenomena in the development and progress of a number of human tumors of the urothelium.