Expression screening of a yeast artificial chromosome contig refines the location of the mouse H3a minor histocompatibility antigen gene

The H3 complex, on mouse Chromosome 2, is an important model locus for understanding mechanisms underlying non-self Ag recognition during tissue transplantation rejection between MHC-matched mouse strains. H3a is a minor histocompatibility Ag gene, located within H3, that encodes a polymorphic peptide alloantigen recognized by cytolytic T cells. Other genes within the complex include beta2-microglobulin and H3b. A yeast artificial chromosome (YAC) contig is described that spans the interval between D2Mit444 and D2Mit17, a region known to contain H3a. This contig refines the position of many genes and anonymous loci. In addition, 23 new sequence-tagged sites are described that further increase the genetic resolution surrounding H3a. A novel assay was developed to determine the location of H3a within the contig. Representative YACs were modified by retrofitting with a mammalian selectable marker, and then introduced by spheroplast fusion into mouse L cells. YAC-containing L cells were screened for the expression of the YAC-encoded H3a(a) Ag by using them as targets in a cell-mediated lympholysis assay with H3a(a)-specific CTLs. A single YAC carrying H3a was identified. Based on the location of this YAC within the contig, many candidate genes can be eliminated. The data position H3a between Tyro3 and Epb4.2, in close proximity to Capn3. These studies illustrate how genetic and genomic information can be exploited toward identifying genes encoding not only histocompatibility Ags, but also any autoantigen recognized by T cells.     

Rapid multiplex analysis for the factor V Leiden and prothrombin G20210A mutations associated with hereditary thrombophilia

Thromboembolic episodes are common events and affect approximately one in 1,000 persons annually. Pulmonary embolism alone accounts for 50,000 to 100,000 deaths per year in the United States with > 50% of those being elderly persons. Resistance to activated protein C is the most common inherited disorder associated with hereditary thrombophilia. A missense mutation has been identified in the gene coding for coagulation factor V (codon 506) which renders this procoagulant factor resistant to inactivation by activated protein C resulting in an increased risk for venous thrombosis. Recently, a second polymorphism was identified in the prothrombin gene (factor II) which is also associated with increased risk for venous thrombosis. Because of the high prevalence of these two mutations in the general population as well as in specific patient populations, the ability readily to detect these two mutations must be feasible. In this study, we evaluated 303 patients for the prothrombin mutatin (G20210A) which were previously tested for the factor V mutation using established polymerase chain reaction-mediated restriction fragment length polymorphism assays. In these patients, 30 (9.9%) were found to be heterozygous for the factor V Leiden mutation with no homozygous mutants identified. Twenty individuals (6.6%) were heterozygous for the prothrombin G20210A mutation, and we identified two individuals (0.66%) who were homozygous for the 20210A allele. Of the total 303 individuals screened, two were double heterozygotes for both the factor V Leiden and the prothrombin gene mutations. We also describe a multiplex polymerase chain reaction-mediated restriction fragment length polymorphism assay for detecting both mutations in a single-tube double-enzyme digestion reaction making identification of these two mutations easily achievable.     

Synthesis and biological activities of phenyl piperazine-based peptidomimetic growth hormone secretagogues

A new class of potent, orally active phenyl piperazine-based GH secretagogues have been discovered from attempts to mimic the arrangement of the phenyl substituent in the spiroindanyl piperidine and spiroindoline sulfonamide privileged structures of 4 and 1, respectively. The best of these compounds, 18 (EC50 = 2.8 nM) is nearly as potent as MK-0677 for releasing GH from rat pituitary cells.     

Prenatal diagnosis of trisomy 13 on fetal cells obtained from maternal blood after minor enrichment

In a pilot study to establish fetal nucleated red blood cell (NRBC) detection in maternal blood, trisomy 13 was diagnosed by FISH analysis at 11 weeks' gestation. The NRBCs were detected after a single-step ficoll density gradient enrichment. In blood samples taken both before and after CVS, 52 and 80 NRBCs, respectively, were found to be positive for fetal haemoglobin. In 47 per cent of these cells, FISH analysis for X and Y chromosomes confirmed the fetal sex. Moreover, 48 per cent of these NRBCs showed three fluorescent signals for a chromosome 13 probe, which confirmed the diagnosis of trisomy 13, previously detected at CVS karyotyping. This is the first report of non-invasive prenatal diagnosis of trisomy 13, i.e., pre-CVS, in the first trimester. The high number of fetal NRBCs detected indicates a connection with aneuploidy, probably due to early impairment of the feto-maternal barrier.     

Insulin-like growth factor binding protein-1 at mouse neuromuscular synapses

The insulin-like growth factor (IGF) signaling system includes the growth factors and their cell surface receptors, along with circulating IGF binding proteins (IGFBPs) that may alter and modulate the action of these neurotrophic hormones. These IGFBPs, along with IGFs and receptors, have been detected in various tissues including the brain. In this study, using polyclonal antibody to human IGFBP-1 or bovine IGFBP-2, we found that mouse muscle extracts contain similar-sized proteins that cross-react with these antibodies on Western immunoblots. After establishing that these antibodies reacted with the homologous murine IGFBPs, we performed immunocytochemistry to demonstrate the localization of IGFBP-1 at the neuromuscular junction, a model nicotinic, cholinergic synapse, as well as within intramuscular nerves. IGFBP-2, a distinct macromolecule, is present on the surface of muscle fibers and is not present within synapses or nerves.     

Antimetabolite-induced increases in the invasive capacity of murine leukaemia L1210 cells

Pretreatment of murine leukaemia L1210 cells with non-lethal concentrations of various antimetabolites increased the in vitro invasive capacity of these cells into monolayers of rat embryo fibroblasts. The increase in invasive capacity was partly correlated with the induced cell cycle arrest. The concomitant increase in cell surface fucosylation and inhibition of invasion with sulphate indicate a role for glycoproteins in this process. Our results suggest that treatment with antimetabolites may lead to a more aggressive phenotype by altering cell surface properties.     

Process Requirements for Cost-Effective Precision Grinding

Costs in precision cylindrical grinding are compared for different abrasives, machines and grinding conditions. The analysis is for repeated batch production. Account is taken of machine cost and abrasive cost. Cost comparisons were based on extensive trials to assess re-dress life against workpiece quality requirements. Experiments show that different workpiece materials require different strategies to reduce costs. Easy-to-grind AISI 52100 and difficult-to-grind Inconel 718 materials were ground at conventional speeds and at high speeds. It is shown that wheel speed affects production rate through acceptable values of re-dress life, removal rate and dwell time. Advantages were gained using vitrified CBN at conventional speed and at high speed. For both materials, vitrified CBN wheels used at high speed, gave better quality at lower cost than conventional abrasives. Wheel costs became negligible and labour costs greatly reduced. Re-dress life trials, usually neglected, are shown to be essential to reduce costs and maintain quality [1].     

The inverse oxidation phenomenon

The oxidation rate of some “as manufactured” graphites increases with increased distance from the surface. The preponderance of the data supports the conclusion that this phenomenon is due to increased catalytic activity of one or more of the impurities in the graphite, and that the increase in catalytic activity is due to changes in the chemical state of the impurities.