Differing populations of intracisternal A-particle genes in myeloma tumors and mouse subspecies.

Intracisternal A-particle genes form a family of endogenous retrovirus-like genetic elements that are transcribed in mouse plasmacytomas (myeloma tumors). Two types of A-particle genes that can be differentiated by a sequence of 0.5 kilobase found in one type but not the other have been identified. Quantitative Southern blot analysis was used to measure the populations of different A-particle genes in DNAs from BALB/c mice, the Japanese subspecies Mus musculus subsp. molossinus, and myeloma tumors. The majority of the genes (715 copies per haploid genome or 76%) were found to be nearly identical except for small changes in conserved restriction enzyme sites. The second type of A-particle gene was much less abundant with 90 copies representing approximately 10%. The A-particle RNA in MOPC104E and MOPC315 was found to be colinear with a small portion of this latter type, comprising only 2% of the endogenous intracisternal A-particle sequences. Myeloma tumor DNA was found to have a two- to fourfold increase in the number of these genes, suggesting that the intracellular viruses have been activated to produce a double-stranded complementary DNA which subsequently integrated into the tumor genome. Analysis of M. musculus subsp. molossinus DNA revealed similar but shifted populations of A-particle genes, when compared with BALB/c DNA, except for the absence of a prominent EcoRI-HindIII band at 3.9 kilobases. This latter band, representing approximately 15% of the A-particle genes in BALB/c DNA, was shown to be a deletion variant of the most abundant gene family.     

Construction and Characterization of Viable Deletion Mutants of Simian Virus 40 Lacking Sequences near the 3′ End of the Early Region

Five viable deletion mutants of simian virus 40 (SV40) were prepared and characterized. These mutants lack 15 to 60 base pairs between map positions 0.198 and 0.218, near the 3′ end of the early region of SV40 and extend further into the body of the A gene, encoding the large T antigen, than previously described deletion mutants. These mutants were isolated after transfection of monkey kidney CV-1p cells with full-sized linear DNA prepared by partial digestion of form I SV40 DNA with restriction endonucleases HinfI or MboII, followed by removal of approximately 25 base pairs of DNA from the 5′ termini using λ-5′-exonuclease and purification of the DNA in agarose gels. Based on camparisons of the DNA sequence of SV40 and polyoma virus, these mutations map in the 19% of the SV40 A gene that shares no homology with the A gene of polyoma virus. The mutations exist in two different genetic backgrounds: the original set of mutants (dl2401 through dl2405) was prepared, using as a parent SV40 mutant dl862, which has a deletion at the single HpaII site (0.725 map unit). A second set (dl2491 through dl2495) contains the same deletions in a wild-type SV40 (strain SV-S) background. Relative to wild-type SV40, the original mutants showed reduced rates of growth, lower yields of progeny virus and viral DNA, and smaller plaque size; in these properties the mutants resembled parental dl862, although mutant progeny yields were usually lower than yields of dl862, suggesting a possible interaction between the two deletions. The second set of mutants had growth properties and progeny yields similar to those of wild-type SV40; however, Southern blotting experiments indicated that viral DNA replication proceeds at a slightly reduced rate. All of the mutants transformed mouse NIH/3T3 cells and mouse embryo fibroblasts at the same frequency as wild-type SV40. Mutants dl2402, dl2492, and dl2405 consistently produced denser and larger foci in both types of cells. All mutants directed the synthesis of shortened large T antigens. Adenovirus helper function was retained by all mutants.     

A test for hormonal responsiveness in a mammary epithelial cell line, NMuMg

The NMuMG cell line derived from normal mouse mammary epithelial cells was tested for responsiveness to hormones. The hormones studied included insulin, glucocorticoids (cortisol and dexamethasone), and prolactin. In addition to membrane bound insulin receptors and prolactin receptors, the cells had 2 X 10(4) cytoplasmic glucocorticoid receptors per cell. Morphological changes were observed in response to hormones. Clusters of cells appeared with greatly increased diameter, and the number of cells per plate was reduced. The rate of DNA synthesis, corrected by cell number, indicates that cell division, and hence cell turnover, was increased by the combination of all three hormones. Insulin greatly enhanced protein synthesis, but glucocorticoid and prolactin did not further increase the rate. The combination of three hormones produced a change in the synthesis of histones, consistent with the increase in cell turnover. There were substantial responses of enzyme activities to hormonal treatment of the cells. Insulin by itself induced a doubling of the activity of glyceraldehyde phosphate dehydrogenase and perhaps a modest increase in NADH-cytochrome c reductase. Lactose synthetase activity showed a three- to fourfold induction of both A and B subunits of the enzyme when the cells were treated with insulin, glucocorticoid, and prolactin, and the effect of the latter two hormones was shown to be additional to that of insulin.     

Non-correlation of phosphorylation of the P-light chain and the actin activation of the ATPase of chicken gizzard myosin.

1. The enzymic properties of myosin isolated from chicken gizzard by three different methods have been compared. 2. Although the specific Ca2+-stimulated ATPases of all preparations were similar and high, there were significant differences in the specific activities of the Mg2+-stimulated actomyosin ATPases. 3. There was no direct correlation between the Mg2+-stimulated actomyosin ATPase activity and the extent of P-light-chain phosphorylation in any of the three myosin preparations. 4. A fraction that activates the Mg2+-stimulated actomyosin ATPase of gizzard muscle has been isolated from a gizzard muscle filament preparation. 5. The activator was specific for the Mg2+-activated actomyosin ATPase of smooth muscle. 6. The activator required the addition of calmodulin for full effect.     

Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. III. Ir gene control of lymphocyte transformation correlates with binding of the mitogen to specific Ia-bearing cells

The supernatant from Mycoplasma arthritidis broth cultures (MAS) contains a T cell mitogen that is under Ir gene control. Responsiveness to this mitogen is dictated by the I-E/I-C subregion of the major histocompatibility complex and is dependent upon adherent radioresistant Ia+ accessory cells from responding haplotype animals. In this study, we established that MAS could be removed from culture supernatants by absorption with spleen cells from mice that themselves are responsive to the mitogen (k and d haplotypes), but activity is not removed by spleen cells from mouse strains that are nonresponsive to the mitogen (b, q, and s haplotypes). Absorption studies with lymphoid cells from congenic and recombinant strain mice established that absorption of the mitogen was itself linked to the I-E/I-C subregion of the major histocompatibility complex. Thymocytes from responding haplotype strains were incapable of removing MAS activity, and spleen cells devoid of Thy-1-positive cells retained their full absorbing capacity. The ability to effectively absorb MAS activity was abrogated by the pretreatment of spleen cells with anti-Ia antiserum and complement. Furthermore, the ability of spleen cells from responding haplotype strains to respond to MAS was blocked by the addition of anti-Ia serum to the cell cultures. Whereas the latter treatment resulted in an almost complete elimination of MAS responsiveness, the ability of similarly treated spleen cells to respond to the mitogens PHA and Con A was only minimally depressed. These results are consistent with our hypothesis that the mitogenic moiety of MAS actually binds to I-E/I-C-coded Ia antigens.     

Structural analysis of a highly acetylated protein using a curved-field reflectron mass spectrometer

Matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry (MS/MS) were used to determine the multiple acetylation sites in the histone acetyltransferase (HAT): p300-HAT. Partial cleavage of the peptides containing acetylated lysine residues by trypsin provided a set of nested sequences that enabled us to determine that multiple acetylation occurs on the same molecule. At the same time, cleavages resulting in a terminal unacetylated lysine suggested that not all of these sites are fully modified. Using MS and MS/MS, we were able to characterize both the unmodified and acetylated tryptic peptides covering more than 82% of the protein.     

Activity of alpha- and theta-defensins against primary isolates of HIV-1

Theta-defensins are lectin-like, cyclic octadecapeptides found in the leukocytes of nonhuman primates. They are also homologues of the more familiar alpha-defensins expressed by humans and certain other mammals. This study compares the ability of six theta-defensins (hominid retrocyclins 1-3 and rhesus theta-defensins 1-3) and four human alpha-defensins (human neutrophil peptides (HNPs) 1-4) to bind gp120 and CD4. In addition, we compared the ability of these theta-defensins and HNP-1 to protect J53-BL cells (an indicator cell line) from primary HIV-1 isolates that varied in subtype and coreceptor usage. The most potent theta-defensin, retrocyclin-2, bound with exceptionally high affinity to gp120 (K(D), 9.4 nM) and CD4 (K(D), 6.87 nM), and its effectiveness against subtype B isolates (IC(50), 1.05 +/- 0.28 microg/ml; 520 +/- 139 nM) was approximately twice as great as that of HNP-1 on a molar basis. We also show, for the first time, that human alpha-defensins, HNPs 1-3, are lectins that bind with relatively high affinity to gp120 (K(D) range, 15.8-52.8 nM) and CD4 (K(D) range, 8.0-34.9 nM). Proteins found in human and FBS bound exogenous HNP-2 and retrocyclin-1, and competed with their ability to bind gp120. However, even the low concentrations of alpha-defensins found in normal human serum suffice to bind over half of the gp120 spikes on HIV-1 and a higher percentage of cell surface CD4 molecules. Although this report principally concerns the relationship between carbohydrate-binding and the antiviral properties of alpha- and theta-defensins, the lectin-like behavior of defensins may contribute to many other activities of these multifunctional peptides.     

Rapid annexin-V labeling in synaptosomes

Distal neuronal terminals may be the site of apoptotic events and early synapse loss in neurodegenerative disease. To examine apoptosis in synaptic regions, we established a cell-free assay using a rat brain crude synaptosomal preparation (P-2 fraction) as a model system. The apoptosis marker annexin-V was used to measure phosphatidylserine (PS) exposure, and to ensure that only intact terminals were assayed, synaptosomes were dual labeled with a viability marker (calcein AM). Fluorescence was quantified by flow cytometry analysis. Annexin-V labeling increased rapidly in synaptosomes, following a 1 min incubation with staurosporine. However, increased caspase-3-like activity was not measured until 30 min with a fluorometric assay. The addition of a peptide inhibitor of caspase-3-like activity (Ac-DEVD-CHO) during homogenization was not able to block the initial increase in annexin labeling, but resulted in a partial blockade of annexin labeling after 30 min. These data demonstrate that PS externalization and caspase activation occur rapidly in this widely used neurochemical preparation.