Thermodynamics of triple helix formation: spectrophotometric studies on the d(A)10.2d(T)10 and d(C+3T4C+3).d(G3A4G3).d(C3T4C3) triple helices.

We have stabilized the d(A)10.2d(T)10 and d(C+LT4C+3).d(G3A4G3).d(C3T4C3) triple helices with either NaCl or MgCl2 at pH 5.5. UV mixing curves demonstrate a 1:2 stoichiometry of purine to pyrimidine strands under the appropriate conditions of pH and ionic strength. Circular dichroic titrations suggest a possible sequence-independent spectral signature for triplex formation. Thermal denaturation profiles indicate the initial loss of the third strand followed by dissociation of the underlying duplex with increasing temperature. Depending on the base sequence and ionic conditions, the binding affinity of the third strand for the duplex at 25 degrees C is two to five orders of magnitude lower than that of the two strands forming the duplex. Thermodynamic parameters for triplex formation were determined for both sequences in the presence of 50 mM MgCl2 and/or 2.0 M NaCl. Hoogsteen base pairs are 0.22-0.64 kcal/mole less stable than Watson-Crick base pairs, depending on ionic conditions and base composition. C+.G and T.A Hoogsteen base pairs appear to have similar stability in the presence of Mg2+ ions at low pH.     

Expression of the glucose transporter isoform GLUT 4 is insufficient to confer insulin-regulatable hexose uptake to cultured muscle cells.

Glucose transporter isoform expression was studied in the skeletal muscle-like cell line, C2C12. Northern and Western blot analysis showed that the insulin-responsive muscle/fat glucose transporter isoform, GLUT 4, was expressed in these cells at very low levels, whereas the erythrocyte isoform, GLUT 1, was expressed at readily detectable levels. Insulin did not stimulate glucose transport in this cultured muscle cell line. The C2C12 cells were then transfected separately with either GLUT 1 or GLUT 4, and stable cell lines expressing high levels of mRNA and protein were isolated. GLUT 1-transfected cells exhibited a 3-fold increase in the amount of the GLUT 1 transporter protein which was accompanied by a 2- to 3-fold increase in the glucose uptake rate. However, despite at least a 10-fold increase in GLUT 4 mRNA and protein detected after GLUT 4 cDNA transfection, the glucose uptake of these cells was unchanged and remained insulin-insensitive. By laser confocal immunofluorescence imaging, it was established that the transfected GLUT 4 protein was localized almost entirely in cytoplasmic compartments. In contrast, the GLUT 1 isoform was detected both at the plasma membrane as well as in intracellular compartments. These results suggest that acute insulin stimulation of glucose transport is not solely dependent on the presence of the insulin receptor and the GLUT 4 protein, and that the presence of some additional protein(s) must be required.     

Insulin-regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations

We have recently described a monoclonal antibody (1F8) that recognizes a form of glucose transporter unique to fat and muscle (James, D. E., Brown, R., Navarro, J., and Pilch, P. F. (1988) Nature 333, 183-185), tissues that respond acutely to insulin by markedly increasing their glucose uptake. Here, we report that rat adipocytes possess two immunologically distinct glucose-transporters: one recognized by 1F8, and one reactive with antibodies raised against the human erythrocyte glucose transporter. Immunoadsorption experiments indicate that these glucose transporters reside in different vesicle populations and that both transporter isoforms translocate from intracellular sites to the plasma membrane in response to insulin. The insulin-regulatable transporter resides in a unique vesicle that comprises 3% or less of the low density microsomes of fat cells and has a limited protein composition that does not include the bulk of another translocatable protein, the insulin-like growth factor II receptor. Immunoprecipitation with 1F8 of microsomal glucose transporters photoaffinity labeled with [3H]cytochalasin B brings down 90% of the label. Similarly, immunoprecipitation with 1F8 of glucose transporters from insulin-stimulated plasma membranes photolabeled with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl-f ors kolin, another transporter-selective reagent, results in 75% of the labeled transporter localized in the immunoprecipitate. Thus, insulin action involves the combined effect of translocation from at least two vesicle pools each containing different glucose transporters. The 1F8-reactive transporter comprises the majority of the total transporter pool that is responsible for the insulin-induced increase in glucose transporter number.     

Conversion of normal human lymphocytes to tumor-specific immunoreactivity by xenogeneic Immune RNA: Blastogenic responses to soluble tumor antigens

Summary Lymphocyte blastogenesis was used as an assay of Immune RNA (I-RNA) activity. Normal, non-immune human lymphocytes following incubation with xenogeneic antitumor I-RNA extracted from the lymphoid organs of specifically immunized sheep underwent blastogenesis when exposed to solubilized human tumor antigens in vitro. Blastogenic responses were, unexpectedly, relatively specific for the tumor type used to immunize the I-RNA donor sheep. No significant blastogenic responses were elicited by the I-RNA extracts or by the antigen preparations themselves. This study suggests that normal, human lymphocytes incubated (sensitized) with I-RNA, in vitro, behave, in terms of antigen recognition, like lymphocytes which have previously been sensitized to tumor antigens and demonstrates that xenogeneic Immune RNA will mediate afferent limb immune responses to human tumor antigens.Supported, in part, by Public Health Service Grant CA-18321 from the National Institute of Health     

Analysis of the rye chromosome constitution and the amount of telomeric heterochromatin in the widely and narrowly adapted hexaploid triticales

Summary Investigations were made on the rye chromosome constitution and on the presence of telomeric heterochromatin in rye chromosomes of the 26 most widely and 24 most narrowly adapted triticale strains. Among widely adapted lines, 22 (85%) had a complete rye genome and four triticales only had chromosomal R-D genome substitutions. Twenty-three (96%) of the 24 most narrowly adapted triticales had substitutions between the chromosomes of the R and D genomes. The most widely adapted triticales accumulated fewer modified rye chromosomes in comparison to narrowly adapted lines. They had from one to three rye chromosomes with heterochromatic deletions: 46% of widely adapted lines had two modified rye chromosomes; 34% had three modified rye chromosomes, and 19% had a single modified rye chromosome. In widely adapted strains, the 1R, 4R, 5R and 6R modified chromosomes were observed; they were present in 80%, 73%, 50% and 11% of the cases, respectively. The most narrowly adapted triticales had from two to four modified rye chromosomes: 58% of the strains had three modified rye chromosomes; 29% had four modified rye chromosomes and 12% had two modified rye chromosomes. The modified 4R and 5R chromosomes were present in all of these lines. The 1R (modified), 6R (modified) and 7R (modified) were found in 83%, 25% and 16%, respectively, of the narrowly adapted strains.Results support the previous observations (Pilch 1980b) that a wide adaptation of hexaploid triticales is associated with the presence of the full potential of rye genome, and that it is independent of the amount of telomeric heterochromatin possessed by rye chromosomes.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Detection of antibodies bound to tumor cell surface antigens with radioiodinated Staphylococcus aureus protein A (SPA)

Summary Staphylococcal protein A (SPA) is known to bind to the Fc portion of certain subclasses of IgG. On the basis of this property, radioiodinated SPA (¹²⁵I-SPA) was used to detect antibodies reacting with surface antigens of tumor cells. Target cells derived from an osteosarcoma growing in C3H/fHeJ mice and antiserum to this tumor prepared in female Hartley guineapigs were used to establish optimum conditions for the assay. Similar optimum conditions were also determined for human melanoma target cells. Target cells were plated at a concentration of either 3×10⁵ cells per well or 1×10⁵ cells per well in Microtest II plates, and allowed to form semiconfluent monolayers for 24–48 h respectively. Target cells thus prepared were treated with antiserum and then with ¹²⁵I-SPA. A minimum of 30 min incubation time was found to be optimal for the antigen-antibody reaction. The quantity of ¹²⁵I-SPA bound to antisera-treated target cells was found to depend on the time of incubation with ¹²⁵I-SPA and on the concentration of SPA used. Longer incubation times and increasing concentrations of SPA resulted in greater amounts of ¹²⁵I-SPA being bound to antiserum treated target cells. This assay was employed for the detection of antibodies in the sera of two melanoma patients and two colon carcinoma patients. The results of absorption analysis suggest that the antibody activity in the sera of the melanoma patients may be of four different specificities: (a) autoantibodies, (b) alloantibodies, (c) antibodies reacting with common, cross-reacting melanoma-associated antigens, and (d) antibodies reacting with unique antigens specific for autologous melanoma cells.     

Palatability and feeding preferences of Uresiphita maorialis (Lepidoptera: Crambidae) for three Sophora species

In a three-hour bioassay, we tested the palatability and feeding preferences of Uresiphita maorialis (kōwhai moth) for Sophora tetraptera, Sophora microphylla and Sophora prostrata. Palatability tests showed no differences among the Sophora species. Feeding preferences, on the other hand, showed that S. tetraptera and S. microphylla leaves are preferred over S. prostrata leaves. Our results support our field observations in Wellington city parks and gardens showing that S. tetraptera and S. microphylla plants frequently have higher densities of larvae than S. prostrata.