Immunoassays and nucleic acid detection with a biosensor based on surface plasmon resonance

A technique based on surface plasmon resonance is described which can be used to detect changes of refractive index that occur when one partner of a molecular binding pair diffuses from solution to bind the other partner which is immobilised on a silver surface. Results for the molecular binding pairs; protein-antibody, hapten-antibody and DNA-DNA are described. Instrumentation necessary for implementation of the technique is detailed. Immunoassay of proteins and haptens is possible in less than one minute with a sensitivity of 10(-9) mol/l. Hybridisation of 10 fmoles of a 97 base target sequence on the 1 mm2 area of detection to an immobilised oligonucleotide probe can be detected in less than five minutes. Advantages of the technique include the ability to record the kinetics of binding reactions in "real time" and the lack of labels in this simple assay format. Methods of improving the sensitivity are discussed.     

The role of the p33:p33/p92 interaction domain in RNA replication and intracellular localization of p33 and p92 proteins of Cucumber necrosis tombusvirus

Replication of plus-stranded RNA viruses is performed by the viral replicase complex, which, together with the viral RNA, must be targeted to intracellular membranes, where replication takes place in membraneous vesicles/spherules. Tombusviruses code for two overlapping replication proteins, the p33 auxiliary protein and the p92 polymerase. Using replication-competent fluorescent protein-tagged p33 of Cucumber necrosis virus (CNV), we determined that two domains affected p33 targeting to peroxisomal membranes in yeast: an N-proximal hydrophobic trans-membrane sequence and the C-proximal p33:p33/p92 interaction domain. On the contrary, only the deletion of the p33:p33/p92 interaction domain, but not the trans-membrane sequence, altered the intracellular targeting of p92 protein in the presence of wt p33 and DI-72(+) RNA. Moreover, unlike p33, p92 lacking the trans-membrane sequence was still functional in supporting the replication of a replicon RNA in yeast, whereas the p33:p33/p92 interaction domain in both p33 and p92 was essential for replication. In addition, p33 was also shown to facilitate the recruitment of the viral RNA to peroxisomal membranes and that p33 is colocalized with (+) and (-)-stranded viral RNAs. Also, FRET and pull-down analyses confirmed that p33 interacts with other p33 molecules in yeast cells. Based on these data, we propose that p33 facilitates the formation of multimolecular complexes, including p33, p92, viral RNA, and unidentified host factors, which are then targeted to the peroxisomal membranes, the sites of CNV replication.     

Is the outcome of in-vitro fertilization and embryo transfer treatment improved by spontaneous or surgical drainage of a hydrosalpinx?

A pilot study was designed to examine whether the outcome of embryo transfer in women with a hydrosalpinx might be improved by surgical drainage of the hydrosalpinx at the time of oocyte collection for in- vitro fertilization treatment. A comparative, controlled but retrospective analysis of the results was performed of all women with infective tubal damage aged <40 years old, who had ovulatory cycles, a normal uterus and a partner with normal spermatozoa. A standardized treatment regimen was used. A maximum of three embryos were transferred. Hydrosalpinx was defined by prior hysterosalpingography and/or laparoscopy with transcervical dye injection. A total of 237 embryo transfer cycles in women with hydrosalpinges (tubal distension not visible in 151, visible but not drained in 30 and drained in 56) were compared with 705 embryo transfer cycles in women with tubal disease but no hydrosalpinx. Results were analysed in the first three cycles but also separately in the first cycle to check for bias. Success rates were higher in the first cycle, but did not significantly influence overall differences. Implantation rates were significantly reduced overall in the hydrosalpinx group (8.0 versus 13.2% for controls; P < 0.001), being 8.3% (P < 0.01) in the subgroup without evident tubal distension and 7.5% (not significant) in the drained hydrosalpinx group. This study shows that tubal damage with distal occlusion is associated with a marked reduction in embryo implantation, even in the absence of obvious fluid distension. Surgical drainage of distended hydrosalpinges appears to offer no benefit.      

The new HLA.

The application of molecular genetics and biochemical techniques to the study of the HLA system over the past few years has resulted in tremendous advances in our knowledge of this area of the human genome. In this article, we describe the "new HLA" that has been pieced together recently through the combined efforts of molecular geneticists, biochemists, and HLA serologists, as well as developments in attempts to elucidate the associations of HLA antigens and disease.     

Relation between high energy phosphate and lethal injury in myocardial ischemia in the dog.

The relationship between progressive depletion of high energy phosphate and the onset of lethal cell injury in ischemic myocardium following coronary occlusion has been evaluated. Myocardial ischemia was induced by proximal occlusion of the circumflex coronary artery for 15, 30, 40, or 60 minutes. Cell injury in the severely ischemic posterior papillary muscle (PP) was evaluated by electron microscopy and by measuring the capacity of slices of the injured PP to maintain electrolytes, resynthesize high energy phosphate, and exclude inulin during in vitro incubation. ATP content in the ischemic myocardium decreased to 35%, 9%, 7%, and 5% of control values after 15, 30, 40, and 60 minutes of ischemia, respectively, and was associated with a corresponding depletion of total adenine nucleotides. The loss of 65% of the ATP after 15 minutes of ischemia (reversible injury) was associated with only minimal ultrastructural changes and no significant defects of electrolytes in incubated slices. However, the depletion of over 90% of the ATP after 40 minutes of ischemia (irreversible injury) was associated with significant fine structural changes and markedly altered cell volume regulation. The results suggest a close relationship between the marked depletion of high energy phsophates and the development of lethal injury in acutely ischemic myocardium.     

Acute inflammatory reaction after myocardial ischemic injury and reperfusion. Development and use of a neutrophil-specific antibody.

Reperfusion of the infarcted canine myocardium after 1 hour of ischemia is associated with an acute inflammatory infiltrate at the border of the infarct. In this paper, we demonstrate that early margination and emigration of neutrophils originate in thin-walled (approximately 5 micrometers) venous cisterns that average 200 micrometers in length and vary from 10 to 70 micrometers in width and show strong constitutive expression of both ICAM-1 and P-selectin; this class of vessels (venous cisterns) appears to be a unique feature in heart. A monoclonal antibody (SG8H6) with specificity for canine neutrophils was developed that allowed much more sensitive immunohistochemical detection of neutrophils in tissue and allowed us to follow tissue infiltration with time. Samples from 1 hour of reperfusion revealed dense margination and substantial emigration of neutrophils associated with the venous cisterns and collecting venules. By 2 hours, there was intense local emigration to the extravascular space between cardiac myocytes. By 3 hours, the infiltrate extended deeper into the infarct, and there was a continuous border zone of neutrophil infiltration that overlapped a region where intact cardiac myocytes strongly expressed ICAM-1 mRNA and extended into the necrotic tissue. At later times, neutrophil migration into infarcted tissue continued to progress. Neutrophil transmigration into reperfused myocardium is more extensive than previously described, and its extravascular distribution during early reperfusion is primarily in the viable border zone of the myocardium where myocyte ICAM-1 mRNA is found. These data are compatible with the hypothesis that extravascular neutrophils may participate in reperfusion injury.     

Nongerminomatous malignant germ cell tumors in children. A review of 89 cases from the Pediatric Oncology Group, 1971-1984

Clinical and morphologic features of 89 cases of childhood yolk sac tumor (YS) and embryonal carcinoma (EC) (29 associated with teratomas) submitted to the Rare Tumor Registry of the Southwest Oncology Group (1971-1979) or the Pediatric Oncology Group (1980-1984) between 1971 and 1984 were reviewed and submitted to statistical analysis. This review showed an improved survival for each 5-year period regardless of tumor site, no statistically significant difference between "pure" tumors and those mixed with other teratomatous components, no statistically significant difference between YS and EC in children, a better than reported prognosis for sacrococcygeal tumors occurring after the neonatal period, a particularly poor prognosis for neonatal "benign" sacrococcygeal teratomas resected without coccygectomy when they recur as YS, excellent survival for all testicular tumors regardless of age or the presence of EC, and the occurrence of mediastinal tumors in females.     

Long-term Impacts and Benefit–Cost Analysis of the Communities That Care Prevention System at Age 23, 12 Years After Baseline

Prevention Science - This study estimated sustained impacts and long-term benefits and costs of the Communities That Care (CTC) prevention system, implemented and evaluated in a longitudinal...