Protein tyrosine phosphatase containing SH2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12-p13.

Protein tyrosine phosphorylation has been implicated in the growth and functional responses of hematopoietic cells. Recently, approaches have been developed to characterize the protein tyrosine phosphatases that may contribute to regulation of protein tyrosine phosphorylation. One novel protein tyrosine phosphatase was expressed predominantly in hematopoietic cells. Hematopoietic cell phosphatase encodes a 68-kDa protein that contains a single phosphatase conserved domain. Unlike other known protein tyrosine phosphatases, hematopoietic cell phosphatase contains two src homology 2 domains. We also cloned the human homolog, which has 95% amino acid sequence identity. Both the murine and human gene products have tyrosine-specific phosphatase activity, and both are expressed predominantly in hematopoietic cells. Importantly, the human gene maps to chromosome 12 region p12-p13. This region is associated with rearrangements in approximately 10% of cases of acute lymphocytic leukemia in children.     

Contrast transfer function of the visual system]

Visually evoked potentials were used to determine the spatial contrast response function of the visual system and the visual acuity of the pigeon. The spatial contrast response describes the relationship between the contrast in a pattern of vertical stripes, whose luminance is a function of position, and the amplitude of the visually evoked response at various spatial frequencies for a given temporal frequency (pattern reversal frequency); it indicates how particular spatial frequencies are attenuated in the visual system. The visually evoked responses were recorded using monopolar stainless steel electrodes inserted into the stratum griseum superficiale of the optic tectum; the depth of penetration was determined on the basis of a stereotactic atlas. The stimulus patterns were generated on a video monitor placed 75 cm in front of the animal's eye perpendicular to the optic axis. The spatial contrast response function measured at 10% contrast and 0.5 Hz reversal frequency shows a peak at a spatial frequency of 0.5 c/deg, corresponding to 1 degree of visual angle, and decreases progressively at higher spatial frequencies. The high-frequency limit (cut-off frequency) for resolution of sinusoidal gratings, estimated from the contrast response function, is 15.5 c/deg, corresponding to a visual acuity of 1.9 min of arc.     

A procedure for biolistic transformation and regeneration of transgenic cotton from meristematic tissue

We have optimized methods for transformation of cotton meristem tissue using the Bio-Rad PDS/1000/He gene gun, selection of transformed tissue, and regeneration of transformed cotton plants. We have used either single or multiple bombardments of cotton tissue with 1.6-Å particles at rupture pressures of 90 or 110 kg/cm². The distance between the tissue and the source of particles can be varied between 3 and 6 cm. After bombardment, transformed cotton tissue is identified by selection for growth on media supplemented with 50 μg/mL kanamycin. Tissue sections that form leaves, shoots and at least two roots are then transferred to media supplemented with 100 mg indoleacetic acid (IAA) to favor formation of extensive root systems. The plantlets are then transferred to soil, hardened off, and grown in the greenhouse. These plants have been confirmed to be transgenic by western-blot analysis of leaf protein extracts with polyclonal antiserum to the neomycin phosphotransferase II gene product.     

Fas activates NF-kappaB and induces apoptosis in T-cell lines by signaling pathways distinct from those induced by TNF-alpha

The p55 tumor necrosis factor (TNF) receptor and the Fas (CD95/APO-1) receptor share an intracellular domain necessary to induce apoptosis, suggesting they utilize common signaling pathways. To define pathways triggered by Fas and TNF-alpha we utilized human CEM-C7 T-cells. As expected, stimulation of either receptor induced apoptosis and TNF-alpha-induced signaling included the activation of NF-kappaB. Surprisingly, Fas-induced signaling also triggered the activation of NF-kappaB in T cells, yet the kinetics of NF-kappaB induction by Fas was markedly delayed. NF-kappaB activation by both pathways was persistent and due to the sequential degradation of IkappaB-alpha and IkappaB-beta. However, the kinetics of IkappaB degradation were different and there were differential effects of protease inhibitors and antioxidants on NF-kappaB activation. Signaling pathways leading to activation of apoptosis were similarly separable and were also independent of NF-kappaB activation. Thus, the Fas and TNF receptors utilize distinct signal transduction pathways in T-cells to induce NF-kappaB and apoptosis.     

Studies of the action of ceramide-like substances (d- andl-PDMP) on sphingolipid glycosyltransferases and purified lactosylceramide synthase

We have studied the effects ofD-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and itsL-enantiomer on glycosphingolipids in cultured normal human kidney proximal tubular cells. We found thatD-PDMP exerted a concentration-dependent reduction in the metabolic labelling and cellular levels of glucosylceramide (GlcCer), lactosylceramide (LacCer), and the globo-series glycosphingolipids, GbOse₃Cer and GbOse₄Cer. It also directly inhibited the activity of UDP-glucose:ceramide 1 4-glucosyltransferase (GlcT-1) and UDP-galactose: GlcCer 1 4 galactosyltransferase (GalT-2). In contrast,L-PDMP had opposite effects on the metabolic labelling of GlcCer, LacCer, and GbOse₃Cer. The levels of GlcCer and LacCer were increased, while the labelling and level of GbOse₄Cer were strongly reduced. Purified GalT-2 from human kidney was inhibited byD-PDMP and stimulated byL-PDMP. It appears likely that the different glycosphingolipid glycosyltransferases possess similar binding sites for the ceramide moiety, which are blocked by binding toD-PDMP and, in the case of GbOse₄Cer synthase, byL-PDMP as well. The stimulatory effects ofL-PDMP on GlcCer and LacCer synthases may be the result of binding to a modulatory site on the glycosyltransferases; in intact cells, the enzyme-analog complex may afford protection against the normal catabolic inactivation of the enzymes.Abbreviations GalT-2 UDP-galactose:GlcCer -galactosyltransferase - GbOse₃Cer Gal1 4Gal1 GlcCer - GbOse₄Cer GalNAc1 3Gal1 4Gal1 GlcCer - GlcCer glucosylceramide - GlcT-1 UDP-glucose:ceramide -glucosyltransferase - GSLs glycosphingolipids - LacCer lactosylceramide - PDMP threo-1-phenyl-2-decanolyamino-3-morpholino-1-propanol     

Transformation of murine bone marrow cells with combined v-raf-v-myc oncogenes yields clonally related mature B cells and macrophages.

Murine bone marrow cells infected with replication-defective retroviruses containing v-raf alone or v-myc alone yielded transformed pre-B cell lines, while a retroviral construct containing both v-raf and v-myc oncogenes produced clonally related populations of mature B cells and mature macrophages. The genealogy of these transformants demonstrates that mature myeloid cells were derived from cells with apparent B-lineage commitment and functional immunoglobulin rearrangements. This system should facilitate studies of developmental relationships in hematopoietic differentiation and analysis of lineage determination.     

Appearance of enzyme activities catalyzing conversion of sterigmatocystin to aflatoxin B1 in late-growth-phase Aspergillus parasiticus cultures

Two activities involved in terminal pathway conversion of sterigmatocystin to aflatoxin B1 were isolated from an aflatoxin-nonproducing mutant of Aspergillus parasiticus (avn-1), and the time course of appearance of the activities in culture was determined. Subcellular fractionation of fungal mycelia resolved the two activities into a postmicrosomal activity which catalyzed conversion of sterigmatocystin to O-methylsterigmatocystin and a microsomal activity which converted O-methylsterigmatocystin to aflatoxin B1. The two activities were absent in 24-h-old cells, increased to optimum levels during the stationary phase, and then declined.     

Characterization of murine A-raf, a new oncogene related to the v-raf oncogene.

A 1.6-kilobase cDNA (A-raf) has been isolated from a murine spleen cDNA library which encodes part of a protein related to the raf oncogene. Its amino acid sequence has 85% homology to raf in a central portion of 100 amino acids. In contrast to raf, A-raf shows a highly restricted tissue distribution of expression, with highest levels observed in epididymis, followed by intestine. When incorporated into a retrovirus, the resulting gag-A-raf fusion gene causes transformation in vitro and induces tumors in newborn mice. Thus, A-raf represents a new proto-oncogene. Transformation by A-raf is independent of ras gene function, as is the case for raf and mos but not other oncogenes.     

Generation of an auto-anti-idiotypic antibody that binds to glucocorticoid receptor

A monoclonal antibody (8G11-C6) that is directed to a region near the ligand-binding site of the glucocorticoid receptor was obtained by an auto-anti-idiotypic route, using a derivative of triamcinolone coupled to thyroglobulin to immunize a mouse. The resulting hybridomas were screened for anti-idiotypic antibody (anti-antisteroid) with Fab fragments of affinity-purified polyclonal rabbit anti-triamcinolone antibody. The anti-idiotypes were then screened for binding to rat cytosol glucocorticoid receptor by a depletion procedure, yielding a clone, 8G11-C6, whose specificity for receptor was verified by sucrose density and Western blot analyses. Depletion was not significantly reduced by prelabeling the cytosol with [3H]triamcinolone acetonide. The anti-idiotype (8G11-C6) bound to Fab fragments of antisteroid and to partially purified receptor in a concentration-dependent manner. Both binding reactions were inhibited only by rabbit serum albumin conjugates of steroids known to bind to the glucocorticoid receptors. Triamcinolone derivatives of lysine and of oligopeptides containing up to six amino acids inhibited the binding of the anti-idiotype to the Fab fragments but not to the receptor, implying that the target epitope of the antisteroid antibody may be closer to its glucocorticoid-binding site than the cross-reacting epitope of the receptor. Our findings demonstrate further the versatility of the auto-anti-idiotypic route for the preparation of anti-receptor antibodies.     

Multiple alpha and beta tubulin genes represent unlinked and dispersed gene families

Cloned cDNA sequences specific for alpha or beta tubulin mRNAs have been used to show that the multigene families which encode either alpha or beta tubulin are unlinked and dispersed throughout the chicken genome. Fractions of chicken chromosomes partially purified by centrifugation on a sucrose gradient were digested with restriction endonucleases and electrophoresed on agarose gels. The DNA was transferred to nitrocellulose filters and hybridized to labeled probes constructed from cloned cDNA sequences specific for alpha or beta tubulin. We find alpha tubulin sequences on four different chicken chromosomes and beta tubulin sequences on at least two different chromosomes. Moreover, using chicken chromosomes further purified with a fluorescent cell sorter, we have been able unambiguously to localize alpha tubulin genes to chromosome 1 and chromosome 8 and two of the beta genes to chromosome 2.