Quadrupole moments of highly deformed structures in the A approximately 135 region: probing the single-particle motion in a rotating potential.

The latest generation gamma-ray detection system, GAMMASPHERE, coupled with the Microball charged-particle detector, has made possible a new class of nuclear lifetime measurement. For the first time differential lifetime measurements free from common systematic errors for over 15 different nuclei ( >30 rotational bands in various isotopes of Ce, Pr, Nd, Pm, and Sm) have been extracted at high spin within a single experiment. This comprehensive study establishes the effective single-particle transition quadrupole moments in the A approximately 135 light rare-earth region. Detailed comparisons are made with theoretical calculations using the self-consistent cranked mean-field theory which convincingly demonstrates the validity of the additivity of single-particle quadrupole moments in this mass region.

Development of a highly quantitative, reproducible assay for determination of chicken T cell growth factor biological activity.

This report examines optimal culture conditions necessary for accurate and sensitive quantification of chicken T Cell Growth Factor (TCGF) activity. With this bioassay, TCGF is quantified by measuring its ability to cause proliferation of splenocytes prestimulated with mitogen. Proliferation is quantified by determining the optical density (OD) or "signal" of test samples in microtiter wells by measuring the incorporation of tetrazolium salt by live cells. To optimize assay conditions, systematic evaluation of the effects of cell culture variables was carried out with the constant aim of increasing signal to noise ratio in the assay. Higher signal to noise ratios were found when using Dulbecco's Modified Eagle's Medium (DMEM) rather than Roswell Park Memorial Institute Medium (RPMI) for basal tissue culture media containing the same supplements. The addition of lipid supplement to the assay system not only increased the proliferation signal, but also decreased the background OD. Incubation temperatures of 41 C rather than 37 C for both the mitogen prestimulation and proliferation phases of the assay also resulted in a higher signal to noise ratio. While incorporating the optimal experimental conditions, a finalized assay procedure employing test sample normalization with an internal assay standard was tested for accuracy. The assay can accurately detect 2 to 15 U/mL of TCGF activity. The within-assay variation ranged from 2 to 13% and the between-assay variation ranged from 11 to 22% depending upon the TCGF preparation being tested. The excellent reproducibility of this assay has facilitated investigations of TCGF production, processing, and purification.

Rapid HLA-DR oligotyping by an enzyme-linked immunosorbent assay performed in microtiter trays.

A simple, sensitive ELISA that is performed in 96-well microtiter plates and that requires less than 90 minutes to complete was developed for HLA-DRB oligotyping. The second exon of HLA-DRB1 was amplified using an unlabeled forward primer and a biotinylated reverse primer and the PCR product was immobilized in avidin-coated wells. Subsequent treatment included exposure to 0.4 N NaOH to remove the nonbiotinylated sense strand, addition of a fluorescein-labeled oligonucleotide probe, one or more 5-minute stringency washes, addition of an alkaline-phosphatase-labeled anti-FL FAB, and then alkaline-phosphatase substrate and amplifier. An intense red-violet color developed within 15 minutes in positive wells and could be quantitated by OD readings at 490-495 nm. To control for stringency and to establish threshold OD values for positive reactions, biotin-labeled antisense oligos that were complementary to the probe or that differed by one or more bases were immobilized in wells in place of PCR products. The assay was sensitive to < 0.05 pmol (approximately 4 ng)/well and required only standard incubators and waterbaths and an optional microplate reader. All reagents were commercially available. The method should facilitate oligotyping of both class I and class II alleles and is adaptable for analysis of other polymorphic gene products.

Indistinguishable nuclear factor binding to functional core sites of the T-cell receptor delta and murine leukemia virus enhancers.

We have previously shown that the delta E3 site is an essential element for transcriptional activation by the human T-cell receptor (TCR) delta enhancer and identified two factors, NF-delta E3A and NF-delta E3C, that bound to overlapping core (TGTGGTTT) and E-box motifs within delta E3. In this study, we show that protein binding to the core motif is necessary but not sufficient for transcriptional activation by the delta E3 element. In contrast, protein binding to the E-box motif does not contribute significantly to enhancer activity. A similar core motif present within the enhancers of T-cell-tropic murine retroviruses has been shown to contribute to transcriptional activity of the viral long terminal repeat in T lymphocytes and to viral T-cell tropism. We therefore determined the relationship between the nuclear factors that bind to the TCR delta and Moloney murine leukemia virus core motifs. On the basis of electrophoretic mobility shift binding and competition studies, biochemical analysis of affinity-labeled DNA-binding proteins, and the binding of a purified core binding factor, the proteins that bound to the TCR delta core site were indistinguishable from those that bound to the murine leukemia virus core site. These data argue that DNA-binding proteins that interact with the core site of murine leukemia virus long terminal repeats and contribute to viral T-cell tropism also play an essential role in the T-cell-specific expression of cellular genes.

Identification of an essential site for transcriptional activation within the human T-cell receptor delta enhancer.

A T-cell-specific transcriptional enhancer was previously identified within the J delta 3-C delta intron of the human T-cell receptor (TCR) delta gene, and seven distinct binding sites for nuclear factors (delta E1 to delta E7) were defined by DNase I footprinting. In this study, we conducted a detailed functional analysis of the various cis-acting DNA sequence elements of the enhancer and show that a 60-bp fragment encompassing delta E3 and delta E4 displays potent enhancer activity, as judged by its ability to activate transcription from the V delta 1 promoter. We show that the interaction of nuclear factors with the delta E3 site is essential for enhancer activity. This element displays significant activity in the absence of additional segments of the enhancer. Further, methylation interference and in vitro mutagenesis identify a site within delta E3 that mediates the binding of two nuclear factors (NF-delta E3A and NF-delta E3C) and that is required for significant transcriptional activation by the enhancer. NF-delta E3C is ubiquitous and may be identical to a previously characterized microE3-binding factor. NF-delta E3A is preferentially expressed in T lymphocytes, and we suggest that this factor may play the dominant role in transcriptional activation through the delta E3 site. This factor interacts with the sequence TGTGGTTT, a motif that is also found within the enhancers of additional TCR and CD3 genes. Nuclear factor binding to delta E4 is also analyzed. One of three specific complexes formed with a delta E4 probe appears to be T-cell specific.

Use of alpha cyclodextrin to eliminate interference by nonesterified fatty acids in calcium measurements.

This study shows that nonesterified fatty acids cause underestimation of serum calcium by some colorimetric methods. The underestimation of calcium occurs infrequently, but can be as large as 20-30 mg/l. Preincubation of the sample with alpha cyclodextrin will eliminate this interference. The procedure is simple, inexpensive, and applicable to most clinical laboratories.