Paper disc estimation of radioactive RNA: studies on the presence and elimination of metabolically generated artifacts from labeled purine and pyrimidine precursors

Conventional usage of the filter-paper disc technique following in vivo hepatic incorporation of certain labeled pyrimidine and purine RNA precursors resulted in the presence of radioactivity on the filter disc in species other than RNA. Thus the filter paper disc technique, conventionally conducted, may be misleading. To eliminate artifacts, it is proposed that replicate filter discs be processed, one group passing through the usual cold TCA washings only, while a parallel group of replicate discs are heated for 20 min at 90°C in 5% TCA to remove RNA. True RNA radioactivity is the difference of counts from the cold TCA precipitable material and the radioactivity remaining after hot TCA extraction.     

Dipetalonema viteae: Extraction and immunogenicity of cuticular antigens from female worms

The surface of adult female Dipetalonema viteae filarial worms was labeled with ¹²⁵I using 1,3,4,6-tetrachloro-3α, 6α-diphenylglycoluril as an iodinating reagent. Ultrastructural autoradiographs showed specific labeling of only the outermost cuticular layers. Surface-labeled worms were homogenized and were extracted with phosphate-buffered saline followed by NaOH. These extracts contained only minor amounts of radioactive TCA-precipitable material. Proteolytic digestion of the remaining sediment either with proteinase K, or thermolysin or subtilisin solubilized about 60% of the radioactive material of which 30% was TCA precipitable. Golden hamsters were injected with proteolytic extracts of unlabeled female worms. Subtilisin and thermolysin extracts provoked antibodies against somatic structures (e.g., gut, uterus, muscles) but not against the cuticle, whereas immunization with the proteinase K extract induced antibodies exclusively against cuticular and hypodermic structures of female and male worms.     

Uptake of liposomes containing the photoprotein obelin by rat isolated adipocytes. Adhesion,endocytosis or fusion?

1. The uptake of liposomes containing the photoprotein obelin by rat isolated adipocytes was investigated with the aim of producing liposome–cell fusion, enabling obelin to be introduced into the cytoplasm of intact cells. 2. Incubation of liposomes containing obelin with rat isolated adipocytes resulted in a time-dependent uptake of entrapped obelin by the adipocytes. The uptake by adipocytes (at 2h) of liposomes prepared from phosphatidylcholine, phosphatidylcholine+phosphatidylserine (molar ratio 4:1) and phosphatidylcholine+N-octadecylamine (molar ratio 4:1) was approx. 6, 10 and 10% of original entrapped obelin per g dry wt. of adipocytes respectively. 3. During incubation with adipocytes some of the liposomes became permeable to Ca²⁺ ions, resulting in stimulation of obelin luminescence from within the liposomes. This increase in permeability to Ca²⁺ seemed to be the result of the interaction of liposomes with the cell membrane. 4. Approx. 50% of liposome uptake could be inhibited by cytochalasin B (500μm). This was consistent with this uptake being the result of endocytosis. The remaining uptake was probably the result of adhesion of liposomes to the cell membrane. 5. In an attempt to detect the presence of cytoplasmic obelin, after incubation of adipocytes with liposomes, a method of causing a rapid rise in cell-membrane permeability to Ca²⁺ was developed in which an anti-cell anti-body–complement reaction occurred at the cell membrane. There was no detectable transfer of active obelin into the cell cytoplasm. 6. After incubation of liposomes with adipocytes in the absence of bovine serum albumin, obelin luminescence from a small proportion of liposomes increased (approx. 1.5%) in response to anti-(5′-nucleotidase) antibody plus complement. 7. It was concluded that under the conditions of these experiments, (a) no detectable transfer (<0.1%) of liposome-entrapped obelin to the adipocyte cytoplasm had occurred, (b) an increase in liposome permeability to Ca²⁺ occurred during incubation with adipocytes, (c) at least 50% of liposome uptake by adipocytes was the result of endocytosis, presumably into secondary lysosomes, and the remaining uptake was apparently the result of loose attachment of liposomes to the cell surface, and (d) in the absence of bovine serum albumin, a portion of at least one surface antigen, the ectoenzyme 5′-nucleotidase, was transferred from the adipocyte membrane to the liposome membrane.     

The involvement of parenchymal,Kupffer and endothelial liver cells in the hepatic uptake of intravenously injected liposomes effects of lanthanum and gadolinium salts

¹²⁵I-labeled albumin or poly(vinyl pyrrolidone) encapsulated in intermediate size multilamellar or unilamellar liposomes with 30–40% of cholesterol were injected intravenously into rats. In other experiments liposomes containing phosphatidyl[Me-¹⁴C]choline were injected. 1 h after injection parenchymal or non-parenchymal cells were isolated. Non-parenchymal cells were separated by elutriation centrifugation into a Kupffer cell fraction and an endothelial cell fraction. From the measurements of radioactivities in the various cell fractions it was concluded that the liposomes are almost exclusively taken up by the Kupffer cells. Endothelial cells did not contribute at all and hepatocytes only to a very low extent to total hepatic uptake of the ¹²⁵I-labels. Of the ¹⁴C-label, which orginates from the phosphatidylcholine moiety of the liposomes, much larger proportions were recovered in the hepatocytes. A time-dependence study suggested that besides the involvement of phosphatidylcholine exchange between liposomes and high density lipoprotein, a process of intercellular transfer of lipid label from Kupffer cells to the hepatocytes may be involved in this phenomenon. Lanthanum or gadolinium salts, which effectively block Kupffer cell activity, failed to accomplish an increase in the fraction of liposomal material recovered in the parenchymal cells. This is compatible with the notion that liposomes of the type used in these experiments have no, or at most very limited, access to the liver parenchyma following their intravenous administration to rats.     

Synthesis and transport of various RNA species in developing embryos of Xenopus laevis.

Embryonic cells of Xenopus laevis were labeled for varying lengths of time, and their nuclear and cytoplasmic RNAs were analyzed, with the following results. (1) The synthesis of small nuclear RNAs (snRNAs) is detected from blastula stage on. (2) The initiation of 4 S and 5 S RNA syntheses occurs at blastula stage. However, while the former is transported into the cytoplasm immediately after its synthesis, the latter remains within the nucleus, until its transport starts later, concomitantly with that of 28 S rRNA. (3) As soon as “blastula” cells start to synthesize 40 S rRNA precursor at 5th hr of cultivation, 18 S rRNA is transported first; the transport of 28 S rRNA begins 2 hr later. (4) On a per-cell basis, poly(A)-RNA is synthesized in blastula stage at a much higher rate than in the later stages. About one-third of the total blastula poly(A)-RNA, and about one-fifth in the case of tailbud cells, is transported quickly into the cytoplasm. Then, it appears that the RNAs which are synthesized at early embryonic stages are transported to the cytoplasm without delays, except for 5 S RNA and snRNAs.     

RNA metabolism of murine leukemia virus: detection of virus-specific RNA sequences in infected and uninfected cells and identification of virus-specific messenger RNA

Virus-specific RNA sequences were detected in mouse cells infected with murine leukemia virus by hybridization with radioactively labeled DNA complementary to Moloney murine leukemia virus RNA. The DNA was synthesized in vitro using the endogenous virion RNA-dependent DNA polymerase and the DNA product was characterized by size and its ability to protect radioactive viral RNA. Virus-specific RNA sequences were found in two lines of leukemia virus-infected cells (JLS-V11 and SCRF 60A) and also in an uninfected line (JLS-V9). Approximately 0.3% of the cytoplasmic RNA in JLS-VII cells was virus-specific and 0.9% of SCRF 60A cell RNA was virus-specific. JLS-V9 cells contained approximately tenfold less virus-specific RNA than infected JLS-VII cells. Moloney leukemia virus DNA completely annealed to JLS-VII or SCRF 60A RNA but only partial annealing was observed with JLS-V9 RNA. This difference is ascribed to non-homologies between the RNA sequences of Moloney virus and the endogenous virus of JLS-V9 cells.Virus-specific RNA was found to exist in infected cells in three major size classes: 60–70 S RNA, 35 S RNA and 20–30 S RNA. The 60–70 S RNA was apparently primarily at the cell surface, since agents which remove material from the cell surface were effective in removing a majority of the 60–70 S RNA. The 35 S and 20–30 S RNA is relatively unaffected by these procedures. Sub-fractionation of the cytoplasm indicated that approximately 35% of the cytoplasmic virus-specific RNA in infected cells is contained in the membrane-bound material. The membrane-bound virus-specific RNA consists of some residual 60–70 S RNA and 35 S RNA, but very little 20–30 S RNA. Virus-specific messenger RNA was identified in polyribosome gradients of infected cell cytoplasm. Messenger RNA was differentiated from other virus-specific RNAs by the criterion that virus-specific messenger RNA must change in sedimentation rate following polyribosome disaggregation. Two procedures for polyribosome disaggregation were used: treatment with EDTA and in vitro incubation of polyribosomes with puromycin in conditions of high ionic strength. As identified by this criterion, the virus-specific messenger RNA appeared to be mostly 35 S RNA. No function for the 20–30 S was determined.     

PROTEIN SYNTHESIS IN ISOLATED NUCLEI FROM ADULT RAT BRAIN

Nuclei from adult rat brains isolated with isotonic sucrose were incubated with [³H]leucine and later purified by centrifugation through hypertonic sucrose solutions. It was found that under these conditions, tritiated leucine was incorporated into TCA precipitable material. Protein synthesis was impaired if the nuclei were treated with the nonionic detergent Triton X-100 or hypertonic sucrose. The presence of puromycin or cycloheximide markedly inhibited the incorporation of the radioactive amino acid. Actinomycin D and RNase did not have any effect on the incorporation. Autoradiography indicated the presence of labelled material within the nuclei and not in cytoplasmic contaminants. Glial nuclei were more actively involved in protein synthesis than neuronal nuclei.     

Identification of the small nuclear RNAs associated with the mitotic chromosomes of Amoeba proteus

Amebas contain 7 electrophoretically distinct species of small nuclear RNAs (snRNAs), some of which are known to associate in a striking manner with mitotic chromosomes. These RNAs can be divided into 2 classes, one consisting of 4 snRNA species that shuttle in a non-random way between nucleus and cytoplasm during interphase and one consisting of 3 snRNA species that do not leave the nucleus at all during interphase. In the work reported here we sought to determine which class is associated with mitotic chromosomes. Through a series of micromanipulative procedures we arranged for the shuttling snRNAs to be the only radioactive molecules in the cell. Such cells were allowed to enter mitosis, whereupon they were fixed and subjected to autoradiography. In those cells no radioactive snRNAs were found associated with mitotic chromosomes. It is concluded, therefore, that those snRNAs that do associate with mitotic chromosomes must be one or more of the non-shuttling species. — In the Discussion, how the non-shuttling snRNAs may function in cell activities is considered.     

A novel experimental approach for systematic identification of box H/ACA snoRNAs from eukaryotes

Box H/ACA snoRNAs represent an abundant group of small non-coding RNAs mainly involved in the pseudouridylation of rRNAs and/or snRNAs in eukaryotes and Archaea. In this study, we describe a novel experimental method for systematic identification of box H/ACA snoRNAs from eukaryotes. In the specialized cDNA libraries constructed by this method with total cellular RNAs from human blood cells, the high efficiency of cloning for diverse box H/ACA snoRNAs was achieved and seven novel species of this snoRNA family were identified from human for the first time. Furthermore, the novel method has been successfully applied for the identification of the box H/ACA snoRNAs from Drosophila and the fission yeast, demonstrating a powerful ability for systematic analysis of box H/ACA snoRNAs in a broad spectrum of eukaryotes.     

Dynamics of annulate lamellae in Drosophila syncytial embryos

The mitotic events in eukaryotic cells are controlled by a family of evolutionary conserved cyclin-dependent kinases (cdk) that phosphorylate cell proteins, which results in the structural reorganization of the entire cell. Our recent studies of Drosophila syncytial embryos have demonstrated that changes in cdk1 activity controlling the assembly and disassembly of nuclear pore complexes also affect the structure of cytoplasmic pores in annulate lamellae. Here, we report a comparative electron microscopic analysis of the dynamics of these organelles during mitosis throughout the development of a Drosophila syncytial embryo. We presume that the distribution of annulate lamellae containing mature cytoplasmic pores across the cytoplasm reflects local reductions in the mitotic kinase cdk1 activity during the development of Drosophila syncytial embryos.     

Mechanisms of insulin degradation by isolated rat adipocytes

Summary We have examined some of the chemical and biological characteristics of the insulin-derived cell-associated radioactivity following incubation of isolated adipocytes with ¹²⁵I-insulin (10^–10 M) for one hour at 37 °C S ephadex G-50 chromatography of the cell-associated radioactivity demonstrated three peaks: peak I eluted with the void volume and consisted of large molecular weight material; peak II comigrated with 1251-insulin; and peak III consisted of small molecular weight degradation products (probably iodotyrosine). When the insulin peak (peak II) was divided into fourths, it was found that the binding and biologic activity of this material was not homogenous; thus, binding and biologic activity (relative to native insulin) fell markedly from the earliest to the latest eluting fractions of this peak. Furthermore, when the entire peak 11 material was applied to DEAE-Sephacel and eluted with a 0.01–0.2 M NaCl gradient, three distinct peaks were observed. These peaks were all 90% TCA precipitable, whereas the ability of the latter two eluting peaks to precipitate with anti-insulin antiserum was markedly reduced. When similar experiments were performed with chloroquine-treated cells, a large increase in cell-associated radioactivity was observed, and Sephadex G-50 chromatography demonstrated that this increase was entirely confined to peaks I and II. When the insulin peak (peak II) was divided into fourths, it was found that chloroquine markedly inhibited the decreased binding and biologic activity, from the earliest to the latest eluting fraction of this peak. Furthermore, when the peak II material (Sephadex G-50) from chloroquine-treated cells was chromatographed on DEAE-Sephacel, this material eluted in a single peak which was 95% TCA precipitable and 106% precipitable by anti-insulin antiserum. In conclusion, these studies demonstrate that: 1) intermediate insulin-derived products with reduced binding and biologic activity are generated in the process of cellular insulin degradation, and 2) the formation of these intermediate products is mediated by a chloroquine-sensitive pathway.     

Expression of recombinant Atlantic salmon serum C-type lectin in Drosophila melanogaster Schneider 2 cells

The Atlantic salmon (Salmo salar) serum lectin (SSL) is a soluble C-type lectin that binds bacteria, including salmon pathogens. This lectin is a cysteine-rich oligomeric protein. Consequently, a Drosophila melanogaster expression system was evaluated for use in expressing SSL. A cDNA encoding SSL was cloned into a vector designed to express it as a fusion protein with a hexahistidine tag, under the control of the Drosophila methallothionein promoter. The resulting construct was stably transfected into Drosophila S2 cells. After CdCl₂ induction, transfected S2 cells secreted recombinant SSL into the cell culture medium. A cell line derived from stably transformed polyclonal cell populations expressing SSL was used for large-scale expression of SSL. Recombinant SSL was purified from the culture medium using a two-step purification scheme involving affinity binding to yeast cells and metal-affinity chromatography. Although yields of SSL were very low, correct folding and functionality of the recombinant SSL purified in this manner was demonstrated by its ability to bind to Aeromonas salmonicida. Therefore, Drosophila S2 cells may be an ideal system for the production of SSL if yields can be increased.     

Effects of 20-OH-ecdysone on Drosophila cells: Regulation of endogenous and transfected genes

Drosophila cell lines respond to physiological doses of 20-OH-ecdysone by entering mitotic arrest and differentiating morphologically. The cells also exhibit changes in gene expression. Several enzyme activities are induced, and the synthesis of cytoplasmic actin and of the four small heat-shock proteins (hsp) is initiated. Hybrid genes, containing the 5′ region of Drosophila heat-shock protein genes ligated to the herpes simplex virus thymidine kinase gene (tk), have been transfected into cells of the Drosophila cell line S3. Constructions containing sequences upstream from hsp 70, or from any of the small hsp genes, show heat-inducible tk expression. Ecdysterone-inducible tk expression is seen only in transfections with small hsp-tk hybrid genes. This transient expression system can be used as an assay for function to define regions of DNA, flanking the coding region of inducible genes, which are necessary for normal gene expression and gene regulation in cultured cells.     

Muscle development in vitro following X irradiation

Myogenic cells obtained from 12-day-old embryonic chicken hind limb and breast muscle were exposed to 5000 rads of X irradiation. Although 10% of the initial cell dissociates were killed by irradiation, the remaining cells were comparable to controls in plating efficiency and light microscopic morphology. Moreover, there was no increase or loss of cells for at least 72 hr in vitro when plated at a density of 2 × 10⁶ cells/60-mm plate. It was found that muscle cell fusion after irradiation proceeded at the same rate and to the same relative extent as in control cultures. Myotubes developed normally; cross-striations were prominent by 5 to 7 days of culture and the cells maintained a well-differentiated state for periods of at least 3 weeks in vitro. In control cultures continuously labeled with 1 μCi/ml of [³H]TdR, 75% of the nuclei within myotubes were heavily labeled by 118 hr; less than 15% of the nuclei within syncytia of irradiated cultures were labeled. Quantitative microphotometry of Feulgen-stained cultures demonstrated that all nuclei within control and irradiated myotubes contained the 2C complement of DNA. Similar experiments conducted with cells released from limbs and breasts of 10-day-old embryos revealed lower absolute levels of cytoplasmic fusion in both control and irradiated samples, however, there was slightly more cell death after exposure to X rays in 10-day-old than 12-day-old material. Nevertheless, considerable cell fusion occurred in irradiated limb and breast cell cultures, consistent with the conclusion that the commitment to myogenesis of prefusion myoblasts is extremely stable even in the face of massive ionizing radiation and that neither cell division nor replication of DNA is an obligatory prerequisite for the in vitro fusion and subsequent differentiation of skeletal muscle obtained from 10- and 12-day-old chick embryos.     

Introduction of enzymes,by means of liposomes,into non-phagocytic human cells in vitro

Liposomes containing entrapped horsedish peroxidase were incubated with three human cell lines in vitro. Although these cells did not ingest latex particles, and took up less than 1 minut of free peroxidase/5 · 10⁶ from the medium, significant amounts (41–164 munits/5 · 10⁶) of peroxidase became cell-associated by 30 min if the enzyme was presented in negatively charged liposomes (phosphatidylchloline/dicetyl phosphate/cholesterol, 70 : 20 : 10 molar ratio). Uptake of liposome-entrapped peroxidase by lymphocytes or fibroblasts was enhanced 2–5-fold if one molar percent of lysophosphatidylcholine was incorporated as a “fusogen”, and was not appreciately diminished by cytochalasin B, an inhibitor of phagocytosis. Lysophosphatidylcholine containing liposomes did not release trapped peroxidase into the medium during incubation, and studies employing the metallochromic dye, arsenazo III demonstrated lack of access of external Ca²⁺ to the internal, enzyme-laden, aqueous compartments; liposome-liposome fusion was also excluded by similar means. Ultrastructural cytochemstry demonstrated peroxidase within liposomes in the free cytosol of cultured cells 15–90 min after apparent liposome-cell fusion. Data provide evidence that multilamellar liposomes can be as vectors for the introjection of missing enzymes into non-phagocytic human cells.     

Drosophila tracheal system formation involves FGF-dependent cell extensions contacting bridge-cells

Development of the ectodermally derived Drosophila tracheal system is based on branch outgrowth and fusion that interconnect metamerically arranged tracheal subunits into a highly stereotyped three-dimensional tubular structure. Recent studies have revealed that this process involves a specialized cell type of mesodermal origin, termed bridge-cell. Single bridge-cells are located between adjacent tracheal subunits and serve as guiding posts for the outgrowing dorsal trunk branches. We show that bridge-cell-approaching tracheal cells form filopodia-like cell extensions, which attach to the bridge-cell surface and are essential for the tracheal subunit interconnection. The results of both dominant-negative and gain-of-function experiments suggest that the formation of cell extensions require Cdc42-mediated Drosophila fibroblast growth factor activity.     

More Sm snRNAs from Vertebrate Cells

There are a number of low-abundance small nuclear RNAs (snRNAs) in eukaryotic cells. Many of them have been assigned functions in the biogenesis of cellular RNAs, such as splicing and 3′ end processing. Here, we present the sequence ofXenopusU12 snRNA and compare the secondary structures of the low-abundance U11 and U12 with those of the high-abundance U1 and U2, respectively. The data suggest functional parallels between these two pairs of snRNAs in pre-mRNA splicing. Using a highly sensitive method, we have identified several new low-abundance snRNAs from HeLa cells. These include five U7 snRNA variants and six novel snRNAs. One of the six novel RNAs is an Sm snRNA, whereas the rest are not immunoprecipitable by either anti-Sm antibodies or anti-trimethylguanosine antibodies. The discovery of these new RNAs suggests that there may be yet more low-abundance snRNAs in the nuclei of eukaryotic cells.