Intracisternal A particles from FLOPC-1 BALB/c myeloma: presence of high-molecular-weight RNA and RNA-dependent DNA polymerase.

Intracisternal A particles from the FLOPC-1 line of BALB/c myeloma have been shown to contain high-molecular-weight RNA (60 to 70S) that is sensitive to RNase, alkali degradation, and heat but resistant to Pronase treatment. The intracisternal A-particle RNA contains tract of poly (A) approximately 180 nucleotides long. As shown in a reconstitution experiment, by antigenic analysis of A-particle preparation and the SC cytopathogenicity assay, the 70S RNA was not due to contamination by type C virus particles. The FLOPC-1 intracisternal A particles also possess an endogenous RNA-dependent DNA polymerase. The enzyme required Mn2+ or Mg2+, dithiothreitol, detergent, and four deoxyribonucleoside triphosphates for maximum activity. Enzymatic activity was maximally stimuated by poly (rC)-oligo (dG)12-18 and less with poly (rG)-oligo (dC)10 or poly (rA)-oligo (dT)12-18 as compare with synthetic DNA/DNA duplex templates such as poly (dA)-oligo (dT)12-18. The enzyme can utilize the A-particle endogenous RNA as template as shown by analysis of the early and late DNA products of the endogenous reaction by CsSO4 isopycnic gradient centrifuation and hybridization of purified 70S or 35S A-particle RNA with the purified complementary DNA product. Approximately 50% of the A-particle complementary DNA also hybridized with oncornavirus RNA.     

Single-chain Tet transregulators

We demonstrate here that the Tet repressor (TetR), a dimeric allosterical regulatory protein, can be converted to a fully functional monomer when connected by a 29 amino acid linker. TetR-based transregulators are widely used to regulate gene expression in eukaryotes. They can be fused to form single-chain (sc) Tet transregulators with two TetR moieties and one eukaryotic regulatory domain. Sc variants of transactivator and transsilencer exhibit the same regulatory properties as their respective dimeric counterparts in human cell lines. In particular, the reverse ‘tet-on’ phenotype of rtTA variants is also present in the sc variants. Coexpression of a reverse transactivator and sc transsilencer leads to reduced background expression and shows full activation upon induction. The data demonstrate that sc Tet transregulators exhibit the phenotype of their respective dimers and lack functional interference when coexpressed in the same cell.     

The bovine leukemia virus encapsidation signal is discontinuous and extends into the 5' end of the gag gene.

In order to define bovine leukemia virus (BLV) sequences required for efficient vector replication, a series of mutations were made in a BLV vector. Testing the replication efficiency of the vectors with a helper virus and helper plasmids allowed for separation of the mutant vectors into three groups. The replication efficiency of the first group was reduced by a factor of 7; these mutants contained deletions in the 5' end of the gag gene. The second group of mutants had replication reduced by a factor of 50 and had deletions including the 5' untranslated leader region. The third group of mutants replicated at levels comparable to those of the parental vector and contained deletions of the 3' end of the gag gene, the pol gene, and the env gene. Analysis of cytoplasmic and virion RNA levels indicated that vector RNA expression was not affected but that the vector RNA encapsidation was less efficient for group 1 and group 2 mutants. Additional mutations revealed two regions important for RNA encapsidation. The first region is a 132-nucleotide-base sequence within the gag gene (nucleotides 1015 to 1147 of the proviral DNA) and facilitates efficient RNA encapsidation in the presence of the second region. The second region includes a 147-nucleotide-base sequence downstream of the primer binding site (nucleotide 551) and near the gag gene start codon (nucleotide 698; gag begins at nucleotide 628) and is essential for RNA encapsidation. We conclude that the encapsidation signal is discontinuous; a primary signal, essential for RNA encapsidation, is largely in the untranslated leader region between the primer binding site and near the gag start codon. A secondary signal, which facilitates efficient RNA encapsidation, is in a 132-nucleotide-base region within the 5' end of the gag gene.     

Multiple Loci within the Major Histocompatibility Complex Confer Risk of Psoriasis

Psoriasis is a common inflammatory skin disease characterized by thickened scaly red plaques. Previously we have performed a genome-wide association study (GWAS) on psoriasis with 1,359 cases and 1,400 controls, which were genotyped for 447,249 SNPs. The most significant finding was for SNP rs12191877, which is in tight linkage disequilibrium with HLA-Cw*0602, the consensus risk allele for psoriasis. However, it is not known whether there are other psoriasis loci within the MHC in addition to HLA-C. In the present study, we searched for additional susceptibility loci within the human leukocyte antigen (HLA) region through in-depth analyses of the GWAS data; then, we followed up our findings in an independent Han Chinese 1,139 psoriasis cases and 1,132 controls. Using the phased CEPH dataset as a reference, we imputed the HLA-Cw*0602 in all samples with high accuracy. The association of the imputed HLA-Cw*0602 dosage with disease was much stronger than that of the most significantly associated SNP, rs12191877. Adjusting for HLA-Cw*0602, there were two remaining association signals: one demonstrated by rs2073048 (p=2×10⁻⁶, OR=0.66), located within c6orf10, a potential downstream effecter of TNF-alpha, and one indicated by rs13437088 (p=9×10⁻⁶, OR=1.3), located 30 kb centromeric of HLA-B and 16 kb telomeric of MICA. When HLA-Cw*0602, rs2073048, and rs13437088 were all included in a logistic regression model, each of them was significantly associated with disease (p=3×10⁻⁴⁷, 6×10⁻⁸, and 3×10⁻⁷, respectively). Both putative loci were also significantly associated in the Han Chinese samples after controlling for the imputed HLA-Cw*0602. A detailed analysis of HLA-B in both populations demonstrated that HLA-B*57 was associated with an increased risk of psoriasis and HLA-B*40 a decreased risk, independently of HLA-Cw*0602 and the C6orf10 locus, suggesting the potential pathogenic involvement of HLA-B. These results demonstrate that there are at least two additional loci within the MHC conferring risk of psoriasis.     

THE INACTIVATION OF BACTERIOPHAGE BY MERCURY BICHLORIDE; THE REACTIVATION OF BICHLORIDE-INACTIVATED PHAGE

1. The inactivation of antistaphylococcus bacteriophage suspended in infusion broth at pH 7.6 and 22°C. by HgCl₂ proceeds according to the equation dP/dt = k [HgCl₂] [P_o – P_i] over the range studied. 2. This inactivation can be reversed by precipitation of Hg⁺⁺ with H₂S. In the present experiments the inactivation was carried out until only some 5 per cent of the initial phage remained active. After reactivation the [P] had increased to 100 per cent of the initial [P].     

Carbon Coated ZnFe2O4 Nanoparticles for Advanced Lithium‐Ion Anodes

The preparation and electrochemical characterization of a new material consisting of carbon coated ZnFe₂O₄ nanoparticles is presented. This material, which offers an interesting combination of alloying and conversion mechanisms, is capable of hosting up to nine equivalents of lithium per unit formula, corresponding to an exceptional specific capacity, higher than 1000 mAh g^?1. Composite electrodes of such a material, prepared using environmentally friendly sodium carboxymethyl cellulose as binder, showed the highest, ever reported, specific capacity and high rate performance upon long‐term testing. Furthermore, in situ X‐ray diffraction analysis allowed identifying the reduction process occurring upon initial lithiation.     

Isolation of two saxitoxin-sensitive sodium channel subtypes from rat brain with distinct biochemical and functional properties

Summary Two different³H-saxitoxin-binding proteins, with distinct biochemical and functional properties, were isolated from rat brain using a combination of anion exchange and lectin affinity chromatography as well as high resolution size exclusion and anion exchange HPLC. The alpha subunits of the binding proteins had different apparent molecular weights on SDS-PAGE (Type A: 235,000; Type B: 260,000). When reconstituted into planar lipid bilayers, the two saxitoxin-binding proteins formed sodium channels with different apparent single-channel conductances in the presence of batrachotoxin (Type A: 22 pS; Type B: 12 pS) and veratridine (Type A: 9 pS; Type B: 5 pS). The subtypes were further distinguished by scorpion (Leiurus quinquestriatus) venom which had different effects on single-channel conductance and gating of veratridine-activated Type A and Type B channels. Scorpion venom caused a 19% increase in single-channel conductance of Type A channels and a 35-mV hyperpolarizing shift in activation. Scropion venom double the single-channel conductance of Type B channels and shifted activation by at least 85 mV.     

Peripheral-type benzodiazepine receptors mediate translocation of cholesterol from outer to inner mitochondrial membranes in adrenocortical cells

In previous studies we demonstrated that peripheral-type benzodiazepine receptors (PBR) were coupled to steroidogenesis in several adrenocortical and Leydig cell systems (Mukhin, A.G., Papadopoulos, V., Costa, E., and Krueger, K.E. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9813-9816; Papadopoulos, V., Mukhin, A.G., Costa, E., and Krueger, K.E. (1990) J. Biol. Chem. 265, 3772-3779). The current study elucidates the specific step in the steroid biosynthetic pathway by which PBR mediate the stimulation in steroid hormone production. The adrenocorticotropin (ACTH)-responsive Y-1 mouse adrenocortical cell line was used to compare the mechanisms by which ACTH and PK 11195 (a PBR ligand) stimulate steroidogenesis. The effects of these agents were studied at three stages along the steroid biosynthetic pathway: 1) secretion of 20 alpha OH-progesterone by Y-1 cell cultures; 2) pregnenolone production by isolated mitochondrial fractions; 3) quantities of cholesterol resident in outer and inner mitochondrial membrane fractions. Steroid synthesis stimulated by ACTH was blocked by cycloheximide, an effect documented by other laboratories characterized by an accumulation of mitochondrial cholesterol specifically in the outer membrane. In contrast, PK 11195-stimulated steroidogenesis was not inhibited by cycloheximide, and the magnitude of the stimulation was markedly enhanced when the cells were pretreated with cycloheximide and ACTH. When isolated mitochondria were used, stimulation of pregnenolone production by PK 11195 was largely independent of exogenously supplied cholesterol, indicating that PBR act on cholesterol already situated within the mitochondrial membranes. This phenomenon was found to be the result of a translocation of cholesterol from outer to inner mitochondrial membranes induced by the PBR ligand. These studies therefore suggest that mitochondrial intermembrane cholesterol transport in steroidogenic cells is mediated by a mechanism coupled to PBR.     

Application of a unique monoclonal antibody as a marker for nonproliferating subpopulations of cells of some tissue

A monoclonal antibody (clone S-30), directed to a protein of 57,000 daltons, was developed from the fusion of mouse myeloma cells and the spleen cells of mice injected with cytoskeletal extracts of fibroblasts that have been aged in in vitro culturing conditions according to a schedule of serial passaging (Cristofalo VJ, Charpentier R: J Tissue Culture Meth 6:117, 1981; Wang E: J Cell Biol, submitted). The staining activity of S-30 antibody was observed exclusively in the nuclei of nonproliferating senescent fibroblasts, but not in their young counterparts. Immunolocalization of S-30 antibody in frozen tissues from various sites reveals the positive staining reaction in the nuclear envelope region in those cells that are at the final stage of differentiation and are no longer replicating. These tissue sites include epithelial cells of the suprabasal layer of epidermis, hair sheath, and tongue, a subpopulation of fibroblasts in the dermis, chondrocytes, hepatocytes, and cells of cardiac muscle. The absence of S-30 staining activity was noted in tissues such as simple epithelium located in the gastrointestinal tract and kidney, and keratinocytes in the basal layer. These results suggest that the S-30 antibody can be used as a marker for nonproliferating cells both in cultured fibroblasts and in some tissues. It seems that the mechanism that controls the cessation of cell proliferation is related, in part, to the postmitotic expression of the 57,000 dalton protein.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.