Mobile Element Insertions Causing Mutations in the Drosophila Suppressor of Sable Locus Occur in Dnase I Hypersensitive Subregions of 5''-Transcribed Nontranslated Sequences

The locations of 16 mobile element insertions causing mutations at the Drosophila suppressor of sable [su(s)] locus were determined by restriction mapping and DNA sequencing of the junction sites. The transposons causing the mutations are: P element (5 alleles), gypsy (3 alleles), 17.6, HMS Beagle, springer, Delta 88, prygun, Stalker, and a new mobile element which was named roamer (2 alleles). Four P element insertions occur in 5' nontranslated leader sequences, while the fifth P element and all 11 non-P elements inserted into the 2053 nucleotide, 5'-most intron that is spliced from the 5' nontranslated leader approximately 100 nucleotides upstream of the translation start. Fifteen of the 16 mobile elements inserted within a approximately 1900 nucleotide region that contains seven 100-200-nucleotide long DNase I-hypersensitive subregions that alternate with DNase I-resistant intervals of similar lengths. The locations of these 15 insertion sites correlate well with the roughly estimated locations of five of the DNase I-hypersensitive subregions. These findings suggest that the features of chromatin structure that accompany gene activation may also make the DNA susceptible to insertion of mobile elements.     

Post-translational modification of a monocyte-specific chemoattractant synthesized by glioma, osteosarcoma, and vascular smooth muscle cells

Chemotaxis is an important step in monocyte recruitment in inflammation, wound healing, and tumor growth. We reported previously that monocyte chemotactic activity secreted by malignant cells and normal smooth muscle cells is associated with a protein or family of proteins that are related to the monocyte-specific smooth muscle cell-derived chemotactic factor (SMC-CF) (Graves, D. T., Jiang, Y. L., Williamson, M. J., and Valente, A. J. (1989) Science 245, 1490-1493). Similar monocyte chemotactic proteins (MCP-1) produced by U-105MG human glioma cells have also been identified (Yoshimura, T., Robinson, E. A., Tanaka, S., Appella, E., Kuratsu, J., and Leonard, E. J. (1989) J. Exp. Med. 169, 1449-1459). We now report that the MCP-1 gene is expressed in MG-63 human osteosarcoma and vascular smooth muscle cells and that SMC-CF antiserum specifically immunoprecipitates proteins synthesized by U-105MG glioma cells. Experiments were undertaken to elucidate the processing pathway of MCP-1/SMC-CF-like proteins in each of these cell types. These experiments demonstrate that larger MCP-1/SMC-CF-like proteins are derived from a Mr = 9000 precursor. Post-translational modification involves the addition of O-linked carbohydrates and sialic acid residues. Differences in carbohydrate processing account for the heterogeneity in MCP-1/SMC-CF-like proteins produced by different cell types. Secretion of these proteins occurs rapidly following processing events in the endoplasmic reticulum-Golgi compartment.     

Kinetic analysis of the separate phosphorylation events in the phosphorylase kinase reaction

Glycogen phosphorylase, a dimer of identical subunits, is activated by phosphorylase kinase-catalyzed phosphorylation of one serine residue in each subunit. In this paper, the effect of the phosphorylation of one subunit on the phosphorylation of the other subunit was examined. The three forms of phosphorylase, phosphorylase b (nonphosphorylated), phosphorylase ab (one subunit phosphorylated), and phosphorylase a (both subunits phosphorylated), were separated by anion-exchange high-performance liquid chromatography (HPLC). Purified phosphorylase ab was found to be stable under the conditions of the phosphorylase kinase assay. Initial rate kinetics showed that phosphorylase kinase had a lower KM for phosphorylase ab (3.9 +/- 0.24 microM) than for phosphorylase b (14.9 +/- 2.6 microM). Using the HPLC separation as a simultaneous assay for the three forms of phosphorylase during the phosphorylase kinase reaction, it was found that the pseudo-first-order rate constant for the second phosphorylation step (k2) was 3.7 times greater than that for the first step (k1). The activator AMP reduced the ratio k2/k1 from 3.7 without AMP to 1.4. When the monomeric gamma delta complex of phosphorylase kinase subunits was used as the enzyme, the ratio k2/k1 was 2.1, compared to 3.7 with the multimeric holophosphorylase kinase. One explanation for these data is that phosphorylation of one subunit of phosphorylase b causes conformational changes that make the other subunit a better substrate for the kinase. In this context, the effect of AMP is to reduce the conformational differences between phosphorylases b and ab, and the gamma delta complex is less sensitive to the conformational differences between the two forms of phosphorylase.     

A rationale for the design of an inhibitor of tyrosyl kinase

Two gastrin analogs containing a D- and a L-tetrafluorinated tyrosyl residue (Arg-Arg-Leu-Glu-Glu-Glu-Glu-Glu-Ala-(F4)Tyr-Gly) were synthesized and tested as substrates and inhibitors of the insulin receptor kinase. No phosphorylation of these peptides was observed, but both gastrin analogs were effective inhibitors in the microM range. Although the D- and L-tetrafluorotyrosine-gastrin analogs differ in the sequence by only 1 amino acid residue, a different inhibitory pattern was obtained with the insulin receptor. The inhibition of all-L-isomer is competitive with respect to both the protein substrate, reduced, S-carboxymethylated, and maleylated lysozyme (RCMM-lysozyme), and ATP with a Ki value of 4 microM. This result corroborates a previous finding (Walker, D. H., Kuppuswamy, D., Visvanathan, A., and Pike, L. J. (1987) Biochemistry 26, 1428-1433) that the kinetic mechanism for insulin receptor is a random Bi Bi mechanism. Different from the L-isomer, the D-analog is competitive to RCMM-lysozyme and noncompetitive toward ATP and gives an apparent inhibition constant of 20 microM. A free tetrafluorotyrosine also shows a competitive inhibition to protein substrate, RCMM-lysozyme (Ki = 18 mM) whereas free tyrosine shows no effect on the activity of insulin receptor. These results show the importance of the charge state and nucleophilicity of the phenolic component in substrate recognition and catalysis and provide a rationale for the design of inhibitors of tyrosyl phosphorylation.     

Epstein-Barr virus in nontumorigenic and tumorigenic nasopharyngeal carcinoma (NPC) somatic cell hybrids

Somatic cell hybrids between mouse fibroblasts and human cells derived from nasopharyngeal carcinoma (NPC) biopsies or NPC tumors propagated in nude mice were examined for the expression of the Epstein-Barr nuclear antigen (EBNA), retention of Epstein-Barr viral (EBV) DNA, and tumorigenicity in nude mice. In all hybrids the expression of EBNA correlated with the detection of EBV-DNA. After more than 2 years in culture, the hybrids examined retained similar amounts of EBV-DNA when compared to previously published data. Retention of EBV-DNA did not correlate with the presence of any particular human chromosome. Use of either rodent cell lines, clone 1D or IT-22, did not affect the retention nor loss of EBV-DNA. For tumorigenicity studies, NPC cells were fused with IT-22 cells and injected into nude mice. Tumor formation did not depend on the presence or absence of EBNA and detectable EBV-DNA sequences; tumorigenicity in these studies could not be correlated with the presence of any particular human chromosome or the origin of the NPC biopsy.     

Early Syncytium Formation by Bovine Leukemia Virus

Bovine leukemia virus (BLV) from either persistently infected bat cells or fetal lamb kidney cells induced rapid syncytium formation in F81 indicator cells. Distinct syncytia were seen within 2 h after inoculation of cells with highly concentrated (500-fold) cell-free BLV preparations and within 4 to 8 h when unconcentrated cell-free BLV preparations were used. Indicator cell densities of 1 x 10(5) to 2 x 10(5) were optimal for rapid and maximal syncytium formation. Pretreatment of BLV with reference BLV leukemic serum and antiserum prepared against purified BLV significantly inhibited (95%) syncytium formation. Reference bovine viral diarrhea virus serum, foamy-like bovine syncytial virus serum, and control serum had little effect (17% inhibition). Antiserum to BLV gp51 inhibited syncytium formation by greater than 96%, whereas antiserum to BLV p24 reduced syncytium activity to a much lesser extent (38% inhibition). Treatment of BLV with beta-propiolactone (0.005 to 0.05%) had little or no effect upon syncytium-forming activity, whereas UV irradiation (15 ergs/mm(2) per s for 30 min) reduced, but did not completely destroy, the fusion activity. However, both beta-propiolactone and UV irradiation drastically reduced the replication potential of BLV, as demonstrated by the lack of p24 expression in the inoculated cells. Concentrations of cycloheximide, cytosine arabinoside, tunicamycin, and 2-deoxy-D-glucose which effectively blocked cellular macromolecular synthesis did not significantly inhibit syncytium formation. These latter results suggested that de novo protein and DNA synthesis as well as protein glycosylation were not required for early syncytium formation. Thus, these experiments demonstrated that replication of BLV by the indicator cells was not essential for cell fusion.     

Evidence for a cerebral cholinergic system and suggested pharmacological patterns of neural organization in the prostomium of the polychaete Nereis virens (SARS)

The histological visualization of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) on frozen sections of prostomia of Nereis virens indicate a concentration of cholinergic activity in the anterior brain. Components are probably sensory epithelial cells with cholinergic axons entering the brain in cephalic nerves and efferent cholinergic axons to prostomial muscle leaving the brain in the same nerves. There are also subepidermal cholinergic cells that may be second order motor neurons serving epidermal mucous cells. The smaller, second lobe of the corpora pedunculata and its associated vertical fibre tract are CAT⁴ and appear continuous, on each side of the cerebral ganglion, with a dorsal and a ventral longitudinal bundle of AChE⁺ fibers. This system tapers to nothing at the level of the posterior eyes. There is a small AChE⁺ component to each optic nerve and AChE is present in the nuchal epithelium. These observations are discussed in relation to earlier studies on aminergic and neurosecretory activity in the same ganglion.     

The human Ia system: Definition and characterization by xenogeneic antisera

The production of xenogeneic anti-Ia serum against Ia antigens in serum has been previously described in the mouse and we now describe the production of xenogeneic anti-human Ia antisera using similar methods. With an indirect resetting technique, Ia-like antibodies were shown to react with the majority of B cells (95%), a subpopulation of T cells, with carbonyl iron adherent cells, and with some E^–Ig^– null cells, but there was no reaction with red cells and platelets. These reactions were the same as those obtained with DRW antisera using cytotoxicity testing. In addition, antigens detected with xenogeneic antisera were also found in serum, where they were found to exist in a low molecular weight, dialyzable form. By the selective removal of different cell surface markers by cocapping, no association could be found with the specifities detected with the xenogeneic anti-Ia antisera and with surface Ig, ₂-microglobulin, or HLA-A and B specificities. Alloantibodies to DRW specificities (but not HLA-A, B specificities) were able to specifically block the binding of the rabbit anti-Ia antibodies to B cells, and reciprocal blocking of rabbit antisera by DRW antibodies was also observed. Several xenogenic antisera were produced by immunizing rabbits with the serum of different individuals. Each antiserum was shown to contain a number of different specificities, as they gave different reaction patterns with different individuals when testing was done both directly and by absorption. These xenogeneic anti-la sera also segregated in a family with HLA-A and B specificities. The detection of a polymorphic antigenic system segregating with the HLA complex, distinct from HLA-A and B specificities, and whose antigens occur predominantly on B cells is therefore described. Because of the similarity of the reactions of the xenogeneic antisera in man to those found in the mouse, and because of the close relationship to the DRW specificities, the system has been provisionally called the H.Ia system.Abbreviations used in this paper AET 2-aminoethyl isothiouronium bromide - ₂-M -2 microglobulin - BSA Bovine serum albumin - H.Ia Human Ia - HuRBC Human red blood cells - Ig Immunoglobulin - Ir Immune response - MHC Major histocompatibility complex - MLR Mixed lymphocyte reaction - NHS Normal human serum - NMS Normal mouse serum - PBL Peripheral blood lymphocytes - PBS Phosphate-buffered saline - RAHIg Rabbit anti-human immunoglobulin - RASIg Rabbit anti-sheep immunoglobulin - RFC Rosette-forming cells - SAHIg Sheep anti-human immunoglobulin - SARIy Sheep anti-rabbit immunoglobulin - SRBC Sheep red blood cells     

Addition of mannose to both the amino- and carboxy-terminal propeptides of type II procollagen occurs without formation of a triple helix

Matrix-free cells obtained from chick embryo cartilage were incubated in the presence of α,α′-dipyridyl and radioactive mannose in order to examine the incorporation of mannose into the propeptide extensions of Type II procollagen. Cell proteins were digested with bacterial collagenase and the digests were examined by polyacrylamide gel electrophoresis. Radioactive mannose was found in fragments from both the N- and C-propeptides, and therefore the results provided the first indication that both these propeptides of Type II procollagen contain mannose. The results also supported previous indications that addition of carbohydrate to the propeptides of procollagen does not require folding of the collagen domain into a triple helix.