RESUMEN

A 5.2 kilobase EcoRI restriction fragment from the Pseudomonas putida HS1 TOL plasmid pDK1, encoding a portion of the lower toluene degradation pathway, was cloned into the E. coli plasmid pBR325. A detailed map of the restriction endonuclease sites was constructed and the nucleotide sequence of three contiguous XhoI fragments, with a combined total length of approximately 3.9 kilobases, has been investigated. This region was determined to contain a total of four separate open reading frames, each preceded by an identical putative ribosome-binding site (nucleotide sequence of 5'-GAGGTG-3'). These open reading frames have been tentatively identified as encoding the lower pathway enzymes catechol 2,3-dioxygenase (C23O) and 1,2-dihydroxycyclohexa-3,5-diene carboxylate dehydrogenase (DHCDH) and a subunit of the toluate 1,2-dioxygenase complex (TO).

Asunto(s)

RESUMEN

Commercial non-lubricated latex condoms were unpackaged and exposed in an environmental chamber to ozone levels (0.3 ppm) commonly present in urban smog conditions. Deterioration was observed by scanning electron microscopy after 18 hours exposure. Loss of mechanical strength was quantitated by measurement of the air pressures necessary to burst the condom and volumes at burst. After 24 hours exposure to ozone the latex surface was covered with craters and after 48 hours the pressure required to burst the condom was 44% that of control samples. Data suggest the need for study of the effectiveness of lubrication and packaging in protecting condoms from environmental factors which may accelerate deterioration.

PIP: Although condoms are subject to tests of strength and leakage, these tests do not predict dependability nor do they consider environmental factors that may increase the probability of breakage. A study of latex condom deterioration was conducted with commercial non-lubricated condoms exposed to ozone levels commonly present in urban smog conditions. Environmental conditions during transport and storage may weaken the latex and contribute to its corrosive effects. In this study, commercial non-lubricated latex condoms were exposed to ozone in an environmental chamber. Deterioration was assessed by scanning electron microscopy after 18 hours of exposure. Condom air burst test protocols from the International Organization for Standardization were used to assess mechanical strength. Results revealed that there was marked deterioration of the condom wall after exposure to ozone after 6-48 hours. Mechanical strength was also decreased after 24 hours' exposure according to air burst test criteria. 3 different brands were tested to ensure that the effects were not limited to 1 brand. The condoms were non-lubricated and unpackaged when exposed, which is important in the interpretation of these results because most condoms marketed in the U.S. are pre-lubricated. Packaging may also provide additional protection. But the study did reveal a fundamental ozone-latex reaction. Anti-ozonant treatments, used in tires and other rubber products, could be an option for the prevention of deterioration in latex condoms. Careful transport and storage of condoms is important to protect them from environmentally accelerated deterioration.

Asunto(s)

Asunto(s)

RESUMEN

Fusion of bovine and goat erythrocytes was studied using the phosphate-calcium protocol. Both bovine and goat red cells are resistant to fusion with phosphate and calcium, under conditions that promote fusion of normal human erythrocytes. Fusion resistance is not related to decreased (5%) membrane deformability of erythrocytes of these species, since chicken erythrocytes which are 40% less deformable than human erythrocytes undergo fusion with efficiency similar to human red blood cells. Incorporation of either phosphatidylcholine or phosphatidylserine into bovine erythrocytes mediated by lipid exchange/transfer protein, caused fusion of these erythrocytes. Fluorescence analysis of merocyanine 540 dye labeled erythrocytes, by flow cytometry, showed that the frequency of cells which exhibit dye binding was much less (35%) in dimyristoylphosphatidylcholine (DMPC) incorporated compared to untreated bovine erythrocytes (80%), indicating that incorporation of DMPC caused closed packing of lipids in the external leaflet of the bilayer. These studies show that fusion of bovine erythrocytes, mediated by phosphate and calcium, has a requirement for either specific phospholipids such as phosphatidylcholine, phosphatidylserine, or closed packing of lipids in the external leaflet of the bilayer.

Asunto(s)

RESUMEN

When a population of human fibrosarcoma HT1080 cells was transfected with a mammalian expression vector and DNA pieces representing either the human whole genome or mouse bulk cDNA, there was a transient increase in the number of adhesive cells in the population. The number of cells with increased adhesion was proportional to the amount of transfected DNA; the increase occurred at a maximal frequency of between 10(-4) and 10(-5) per cell. Increased adhesion in these cells persisted for 9-12 days, corresponding to the period of highly efficient transient transfection, and was accompanied by arrest in cell division. Transfection of non-mammalian DNAs, reduction of transfected mammalian sequence length by restriction enzyme digestion, or omission of expression vector DNA did not permit these shifts in phenotype. The effects seen suggest that expression of specific transfected mammalian DNA sequences suppresses certain phenotypic characteristics in these transformed mammalian cells.

Asunto(s)

RESUMEN

Fusion of normal human, goat, bovine and chicken erythrocytes was investigated using the phosphate-calcium protocol. The relative effects on fusion efficiency of cell shape, membrane deformability, and composition and orientation of phospholipids in the lipid bilayer were ascertained. Exposure of red blood cells to phosphate ions followed by calcium led to cell agglutination at room temperature for all species. Incubation at 37 degrees C for periods up to 2 hours caused hemolysis and membrane-associated protein aggregation followed by fusion in human and chicken erythrocytes but not in bovine or goat red cells. Cell shape may not be a determinant in fusion since bovine, like human erythrocytes, are biconcave but do not fuse with the above protocol. Bovine and goat red blood cells lack phosphatidylcholine in their membranes. Incorporation of phosphatidylcholine into bovine red blood cells, mediated by a specific transfer protein, promoted their fusion. Our results indicate the important role played by phospholipid composition and orientation in red cell membranes for fusion by phosphate and calcium.

Asunto(s)

RESUMEN

Cell selection immediately after DNA-mediated transfection of whole-cell DNA into mammalian cells has been used to select for specific DNA sequences that cause a phenotypic effect. Whole-cell mouse or human DNA was cleaved into a distribution of lengths (0.4-25 kilobase pairs) and transfected into anchorage-independent spontaneously transformed NIH/3T3 cells. Immediately after transaction, anchorage-dependent serum concentration-dependent reverents were selected. The Hirt supernatant, containing extrachromosomal DNA resulting from the transfection, was isolated from the revertants and transfected with high molecular weight carrier DNA into a second population of transformed cells; revertants were again selected. After five to seven cycles of transfection of Hirt supernatant DNA (obtained from revertants selected at the previous cycle) into new populations of transformed cells at each cycle, the reversion frequency had become 5-15 times greater than the spontaneous reversion frequency measured for several subclones of nontransfected or mocktransfected transformed NIH/3T3 cells. When nonmammalian genomic DNAs were used in transfecting a first population of cells, there was no effect on the reversion of frequency even after six cycles of selection. The reversion-enhancing activity of sixth-cycle Hirt supernatant DNA resulting after transfection at the first cycle with mouse or human sequences was destroyed by EcoRI but not by BamHI or Sal I. Sequences resembling human Alu I sequences were found in mouse whole-cell DNA isolated from sixth-cycle revertants generated after transfection of human sequences at the first cycle.

Asunto(s)

RESUMEN

An improved method is described for measuring red cell membrane deformability via aspiration into the 0.6 micron pores of Nuclepore filters. Application of this technique to the membranes of echinocytes formed in various ways showed that the method of preparation, not the final shape, determined deformability. Old and young red cells had equally deformable membranes. Membrane deformability was independent of the internal viscosity of cells suspended in media over the range 300-600 mOsm, while between 300 and 100 mOsm the deformability decreased.

Asunto(s)

RESUMEN

Negative charges on the external surface of red cells were visualized by colloidal iron hydroxide labelling of 50% of the membrane area after osmotic hemolysis and glutaraldehyde fixation. Counts were made over randomly selected areas on electron micrographs at 350,000 x magnification. Statistical analyses showed that at the 95% level of confidence there was no significant difference between oxygenated normal (AA) and sickle (SS) cells in either the distribution or the density of negative charges.

Asunto(s)

RESUMEN

Native DNA from sea urchin embryos contains single-stranded regions (gaps) of up to 3000 nucleotides. The longer gaps (more than 1400 nucleotides) are nonrandomly distributed and are rich in histone gene sequences, other moderately repetitive sequences, and polypyrimidines. The shorter gaps are associated with DNA replication. A method for isolation of the two classes of single-stranded DNA pieces is reported.

Asunto(s)

RESUMEN

The membrane deformability of metabolically depleted human red cells has been measured using a simplified technique of Nuclepore aspiration. Cells depleted in the presence of 25 microM--2 mM Ca2+ were 11.3%-15.7% less deformable than fresh cells. Cells depleted in HEPES-buffered saline with 25 microM Ca2+ and 1 mM EGTA were as deformable as fresh cells. Echinocytes formed by dinitrophenol treatment of fresh cells were also as deformable as fresh cells. Deformability was measured by red cell aspiration in Nucleopore filters with 0.6 micrometer pores and low pore density (2 x 10(6)/cm2). A rapid and reliable separation of cells and filter with little cell disorientation, combined with simultaneous mounting for scanning microscopy, was achieved with the use of double-sided Scotch tape.

Asunto(s)

RESUMEN

Naturally occurring single-stranded regions in native DNA isolated from sea urchin embryos at the morula stage were removed by digestion with S1 nuclease. Renaturation experiments show that this nuclease removes a portion of the repetitive sequences renaturing between Cot 10(-2) and 20 including about two-thirds of the histone genes. The latter was shown by hybridization of S1-treated morula DNA to either sea urchin 9 S polysome RNA (containing histone mRNA) or to Escherichia coli plasmid DNA carrying sea urchin histone genes. Hybridization of sea urchin DNA to strand-separated recombinant histone gene DNA shows that it is the histone gene antisense strand that is missing from the gapped regions of the native DNA from morulae. Present and previous data support the conclusion that a portion of the single-stranded regions are not in random positions in the embryo genome and do not result from the unwinding of DNA in vivo at replication sites.

Asunto(s)

RESUMEN

Incubation of human erythrocytes with either uranyl ions (UO22+) or rare earth metals (La3+, Nd3+, Sm3+, Eu3+, Tb3+, Dy3+ and Yb3+) at 37 degrees C for 30-45 min resulted in the fusion of erythrocytes. Redistribution of membrane-associated particles was observed using colloidal-iron charge labelling and freeze-fracture electron microscopy. The fusion of erythrocytes induced by these agents, unlike Ca2+, did not exhibit the absolute requirement for phosphate. Moreover, agglutination and fusion by these agents was observed in neuraminidase-treated erythrocytes in contrast to Ca2+- and phosphate-induced fusion. Inhibitors of intrinsic transglutaminase activity partially inhibited (35-45%) the fusion induced by UO22+ suggesting that cross-linking of membrane proteins results in protein-free areas of lipid where fusion may be initiated.

Asunto(s)

Asunto(s)

RESUMEN

Previous studies on the genome of Strongylocentrotus purpuratus sea urchin have shown that changes in the nucleotide sequence of inverted repeat sequences occur during embryogenesis. The present study indicates that these sequence changes fail to occur when the embryos are raised in the presence of 5-bromodeoxyuridine. This drug is an analog of thymidine, is incorporated into the DNA during embryogenesis, and inhibits cell differentiation in these embryos.

Asunto(s)

Asunto(s)

RESUMEN

A photoaffinity probe, procaine azide, was employed to determine the sites of interaction of procaine in normal and sickle cell erythrocytes. Studies show that the number of binding sites and affinity of procaine to membranes derived from normal and sickled cell erythrocytes were similar, although procaine retards the in vitro formation of irreversibly sickled cells from cells, The results show that procaine azide, a photoaffinity analogue of procaine, is covalently incorporated into both protein (60--70%) and lipid (40--30%) components of the membrane. Sodium dodecyl sulfate-gel electrophoresis of the labeled ghosts show that procaine binds specifically to band 3 and periodic acid Schiff staining bands in membranes derived from labeled erythrocytes. Binding of procaine or covalent incorporation of procaine azide into membrane proteins does not affect the phosphate transport. Moreover, pre treatment of intact erythrocytes with 4,4-'diisothiocyano-2,2'-stilbene disulfonate, an anion transport inhibitor, did not affect either the binding or covalent incorporationof procaine azide into erythrocytes. These results indicate that the binding of procaine azide to Band 3 protein occurs at a locus different than that involved in anion translocation process.

Asunto(s)

Asunto(s)

RESUMEN

Hairpin-like DNA was prepared in vitro from the family of sequences that are inverted relative to each other and, as pairs, are relatively homologous and adjacent on the sea urchin genome. The majority of these hairpins are shown to have base pair mismatch positions distributed along their stems. Comparison of the hairpins derived from the DNA of morula, blastula, and gastrula stage embryos shows that during embryogenesis there are changes in the average number and position of S1 nuclease-sensitive base pair mismatch sites on the majority of the hairpin stems. Our data indicate that during early embryogenesis there are sequence changes in vivo within the majority of the adjacent inverted repeat sequences of the sea urchin genome. We have also found that there is higher specificity for the occurrence of sequence-change events within that fraction of the inverted repeat sequences that are methylated in vivo.

Asunto(s)

Asunto(s)