The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

Conjugates of folate and anti-T-cell-receptor antibodies specifically target folate-receptor-positive tumor cells for lysis.

High-affinity folate receptors (FRs) are expressed at elevated levels on many human tumors. Bispecific antibodies that bind the FR and the T-cell receptor (TCR) mediate lysis of these tumor cells by cytotoxic T lymphocytes. In this report, conjugates that consist of folate covalently linked to anti-TCR antibodies are shown to be potent in mediating lysis of tumor cells that express either the alpha or beta isoform of the FR. Intact antibodies with an average of five folate per molecule exhibited high affinity for FR+ tumor cells but did not bind to FR- tumor cells. Lysis of FR+ cell lines could be detected at concentrations as low as 1 pM (approximately 0.1 ng/ml), which was 1/1000th the concentration required to detect binding to the FR+ cells. Various FR+ mouse tumor cell lines could be targeted with each of three different anti-TCR antibodies that were tested as conjugates. The antibodies included 1B2, a clonotypic antibody specific for the cytotoxic T cell clone 2C; KJ16, an anti-V beta 8 antibody; and 2C11, an anti-CD3 antibody. These antibodies differ in affinities by up to 100-fold, yet the cytolytic capabilities of the folate/antibody conjugates differed by no more than 10-fold. The reduced size (in comparison with bispecific antibodies) and high affinity of folate conjugates suggest that they may be useful as immunotherapeutic agents in targeting tumors that express folate receptors.

Normal plasma lipoproteins and fertility in gene-targeted mice homozygous for a disruption in the gene encoding very low density lipoprotein receptor.

The very low density lipoprotein (VLDL) receptor is a recently cloned member of the low density lipoprotein (LDL) receptor family that mediates the binding and uptake of VLDL when overexpressed in animal cells. Its sequence is 94% identical in humans and rabbits and 84% identical in humans and chickens, implying a conserved function. Its high level expression in muscle and adipose tissue suggests a role in VLDL triacylglycerol delivery. Mutations in the chicken homologue cause female sterility, owing to impaired VLDL and vitellogenin uptake during egg yolk formation. We used homologous recombination in mouse embryonic stem cells to produce homozygous knockout mice that lack immunodetectable VLDL receptors. Homozygous mice of both sexes were viable and normally fertile. Plasma levels of cholesterol, triacylglycerol, and lipoproteins were normal when the mice were fed normal, high-carbohydrate, or high-fat diets. The sole abnormality detected was a modest decrease in body weight, body mass index, and adipose tissue mass as determined by the weights of epididymal fat pads. We conclude that the VLDL receptor is not required for VLDL clearance from plasma or for ovulation in mice.

Green fluorescent protein as a vital marker and reporter of gene expression in Drosophila.

We have used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a vital marker/reporter in Drosophila melanogaster. Transgenic flies were generated in which GFP was expressed under the transcriptional control of the yeast upstream activating sequence that is recognized by GAL4. These flies were crossed to several GAL4 enhancer trap lines, and expression of GFP was monitored in a variety of tissues during development using confocal microscopy. Here, we show that GFP could be detected in freshly dissected ovaries, imaginal discs, and the larval nervous system without prior fixation or the addition of substrates or antibodies. We also show that expression of GFP could be monitored in intact living embryos and larvae and in cultured egg chambers, allowing us to visualize dynamic changes in gene expression during real time.

Insulin-like growth factor I treatment reduces demyelination and up-regulates gene expression of myelin-related proteins in experimental autoimmune encephalomyelitis.

To compare effects of insulin-like growth factor I (IGF-I) and placebo treatment on lesions that resemble those seen during active demyelination in multiple sclerosis, we induced experimental autoimmune encephalomyelitis in Lewis rats with an emulsion containing guinea pig spinal cord and Freund's adjuvant. On day 12-13, pairs of rats with the same degree of weakness were given either IGF-I or placebo intravenously twice daily for 8 days. After 8 days of placebo or IGF-I (200 micrograms/day or 1 mg/day) treatment, the spinal cord lesions were studied by in situ hybridization and with immunocytochemical and morphological methods. IGF-I produced significant reductions in numbers and areas of demyelinating lesions. These lesions contained axons surrounded by regenerating myelin segments instead of demyelinated axons seen in the placebo-treated rats. Relative mRNA levels for myelin basic protein, proteolipid protein (PLP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase in lesions of IGF-I-treated rats were significantly higher than they were in placebo-treated rats. PLP mRNA-containing oligodendroglia also were more numerous and relative PLP mRNA levels per oligodendrocyte were higher in lesions of IGF-I-treated rats. Finally, a significantly higher proportion of proliferating cells were oligodendroglia-like cells in lesions of IGF-I-treated rats. We think that IGF-I effects on oligodendrocytes, myelin protein synthesis, and myelin regeneration reduced lesion severity and promoted clinical recovery in this experimental autoimmune encephalomyelitis model. These IGF-I actions may also benefit patients with multiple sclerosis.

Primary structure of hepatocyte nuclear factor/forkhead homologue 4 and characterization of gene expression in the developing respiratory and reproductive epithelium.

Members of the winged helix/forkhead family of transcription factors are believed to play a role in cell-specific gene expression. A cDNA encoding a member of this family of proteins, termed hepatocyte nuclear factor/forkhead homologue 4 (HFH-4), has been isolated from rat lung and rat testis cDNA libraries. This cDNA contains an open reading frame of 421 amino acids with a conserved DNA binding domain and several potential transactivating regions. During murine lung development, a single species of HFH-4-specific transcript (2.4 kb long) is first detected precisely at the start of the late pseudoglandular stage (embryonic day 14.5) and, by in situ hybridization, is specifically localized to the proximal pulmonary epithelium. The unique temporal and spatial pattern of HFH-4 gene expression in the developing lung defines this protein as a marker for the initiation of bronchial epithelial cell differentiation and suggests that it may play an important role in cell fate determination during lung development. In addition to expression in the pulmonary epithelium, RNA blot analysis reveals 2.4-kb HFH-4 transcripts in the testis and oviduct. By using mice with genetic defects in spermatogenesis, HFH-4 expression in the testis is found to be associated with the appearance of haploid germ cells and in situ hybridization studies indicate that HFH-4 expression is confined to stages I-VII of spermatogenesis. This pattern of HFH-4 gene expression during the early stages of differentiation of haploid germ cells suggests that HFH-4 may play a role in regulating stage-specific gene expression and cell-fate determination during lung development and in spermatogenesis.

Abatement of hydrocarbons by acid ZSM-5 and BETA zeolites under cold-start conditions

Simulated cold-start tests have been carried out to evaluate the performance of H-ZSM-5 and H-BETA zeolites as hydrocarbon traps under simulated gasoline car exhaust gases, paying special attention to the effect of water on their behaviour. It is concluded that the hydrothermal treatment of the zeolites in the acidic form contributes to the better performance of these materials as hydrocarbon traps since the stabilization of the zeolites takes place. Moreover, the decrease of the surface acidity of the zeolites results in an increase of the Si/Al ratio, which contributes to the decrease of the water affinity for adsorption sites. Thus, the competition with hydrocarbon molecules in the exhaust for the adsorption sites is reduced which increases their trap efficiency. The stabilized H-ZSM-5 is the zeolite that showed the best performance with a propene offset temperature of 240 °C, which should be high enough for the three-way catalyst to carry out its role as catalytic converter.

Development and evaluation of a pollution prevention technology for surface cleaning and decontamination : cooperative research and development agreement with Cold Jet, Inc.

Mode of access: Internet.

Drug Interdiction and Anti-Drug Abuse Act of 1988 [microform] : hearing before a subcommittee of the Committee on Appropriations, United States Senate, One Hundredth Congress, second session, special hearing.

"Fiscal year 1989"--Cover.