Studies on the role of amino acids 38-45 in the expression of a functional thyrotropin receptor.

We previously reported that deletion or substitution of a unique eight-amino acid tract (residues 38-45) in the extracellular domain of the human TSH receptor led to the loss of specific ligand binding to the surface of transfected cells. In the present study we analyzed this region in more detail. Using site-directed mutagenesis of the TSH receptor cDNA, we substituted amino acid residues 38-45, either in three overlapping groups of four amino acids each or individually. The resultant TSH receptor mutant cDNAs were stably transfected into Chinese hamster ovary cells, and the cells were tested for their TSH-binding ability. Our data demonstrate that amino acid residues 38-40 and 42-45 in this region of the human TSH receptor can be substituted without alteration in receptor function and are, therefore, not critical in forming or maintaining the TSH-binding site. However, substitution of Cys41, either alone or together with adjacent amino acids, leads to the loss of TSH binding to its receptor. These data suggest a central role for the amino acid in position 41 in preserving the biological function of the TSH receptor.     

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates

The effect of phorbol esters on the adhesive properties of NIH/3T3 mouse fibroblasts was investigated using plastic substrates precoated with the extracellular matrix proteins fibronectin, collagen, and laminin. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced NIH/3T3 cell attachment to laminin and type IV collagen substrates but had little or no effect on attachment to fibronectin and type I collagen substrates. The effect of PMA in enhancing cell attachment to laminin and type IV collagen substrates was dose dependent between 10(-9) and 10(-7) M. PMA was effective as early as 30 min; the effect reached a maximum at 2 h and decreased gradually. Phorbol 12, 13-dibenzoate and phorbol 12, 13-diacetate were effective but to a lesser extent and phorbol 12-myristate and phorbol 13-acetate showed little or no effect. These results suggest that PMA may enhance NIH/3T3 cell adhesion through effects on laminin and type IV collagen receptors. Retinoic acid, which itself requires at least 6 h to show an effect on attachment, did not have any effect on cell attachment in 2 h and, if anything, slightly inhibited PMA-enhanced cell attachment to laminin and type IV collagen substrates.     

Elutriation of human keratinocytes and melanocytes fromin vitro cultured epithelium

Summary Human epidermal keratinocytes grown in culture and at different stages of differentiation are shown to be viably separated by elutriation. A specific fraction enriched in melanocytes was obtained. Elutriation of cells obtained fromin vitro cultured epithlium could prove useful in studies concerning the biochemistry and molecular markers of cells isolated from normal epithelium and from different pathologies.     

Genetical and biochemical comparisons of alcohol dehydrogenase isozymes from Anastrepha fraterculus and A. obliqua (Diptera: Tephritidae): Evidence for gene duplication

The electrophoretic patterns of the enzyme alcohol dehydrogenase (ADH) from Anastrepha fraterculus and A. obliqua were studied. Two loci were found to code for the enzyme in A. fraterculus, and three in A. obliqua. In both species, all isozymes were active in third-instar larvae. A cationic isozyme (Adh-1) was active mainly in the visceral fat body of both species. In A. fraterculus, the locus had an anionic polymorphic isozyme (Adh-3) that was detected in the parietal fat body. In addition to these two loci, a third locus for an anionic isozyme (Adh-2), which was active in the digestive tube of larvae, was present in A. obliqua and probably resulted from gene duplication. For both species, multiple forms of the isozymes are formed by binding of an NAD-carbonyl compound, as in Drosophila melanogaster. Both larvae and early pupae of A. obliqua had almost twice the specific ADH activity as A. fraterculus. The ethanol content of the host fruit infested with A. obliqua (red mombim) was also higher than that of the host fruit infested with A. fraterculus (guava).This research was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq-PIG 40.2486/82).     

Effect of gamma radiation on resting B lymphocytes. I. Oxygen-dependent damage to the plasma membrane results in increased permeability and cell enlargement

Although the susceptibility of resting B lymphocytes to radiation-induced interphase death is well known, the mechanism by which this occurs is not understood. In this report, we use three measures of plasma membrane integrity (increase in cell volume, uptake of trypan blue, and release of 51Cr) to assess the effect of radiation on the resting B cell plasma membrane. The delivery of 500 to 1000 rad caused the majority of resting B cells to enlarge slightly, whereas 3000 rad caused virtually all of the cells to approximately double in size within 3 to 4 hr. Measurement of the release of 51Cr from resting B cells revealed a similar relationship between the dose of radiation and the loss of radioactive label. Trypan blue exclusion was also found to diminish as a function of radiation dose. An analysis of a variety of lymphoid cells suggested that sensitivity to the membrane damaging effects of gamma radiation was in the order of resting B cells greater than resting T cells greater than a long-term L3T4+ T cell clone greater than a B cell lymphoma. LPS-induced B cell blasts treated with 3000 rad were equivalent to 1000 rad-treated resting B cells. The effects of the gamma radiation could be ameliorated by excluding oxygen (a diradical molecule that can potentially enhance the generation and propagation of highly reactive free radicals) at the time of irradiation, or by adding the free radical scavenging agent cysteamine. These data are compatible with the hypothesis that gamma radiation results in damage to the plasma membrane of resting lymphocytes via the generation of highly reactive free radical species. This damage is reflected in a rapid increase in plasma membrane permeability and swelling of the cells, and may play a major role in causing interphase death.     

Kinetic characterization of reduced pyridine nucleotide dehydrogenases (duroquinone-dependent) in cucurbita microsomes

Stomatal conductances of normally oriented and inverted leaves were measured as light levels (photosynthetic photon flux densities) were increased to determine whether abaxial stomata of Vicia faba leaves were more sensitive to light than adaxial stomata. Light levels were increased over uniform populations of leaves of plants grown in an environmental chamber. Adaxial stomata of inverted leaves reached maximum water vapor conductances at a light level of 60 micromoles per square meter per second, the same light level at which abaxial stomata of normally oriented leaves reached maximum conductances. Abaxial stomata of inverted leaves reached maximum conductances at a light level of 500 micromoles per square meter per second, the same light level at which adaxial stomata of normally oriented leaves reached maximum conductances. Maximum conductances in both normally oriented and inverted leaves were about 200 millimoles per square meter per second for adaxial stomata and 330 millimoles per square meter per second for abaxial stomata. Regardless of whether leaves were normally oriented or inverted, when light levels were increased to values high enough that upper leaf surfaces reached maximum conductances (about 500 micromoles per square meter per second), light levels incident on lower, shaded leaf surfaces were just sufficient (about 60 micromoles per square meter per second) for stomata of those surfaces to reach maximum conductances. This `coordinated' stomatal opening on the separate epidermes resulted in total leaf conductances for normally oriented and inverted leaves that were the same at any given light level. We conclude that stomata in abaxial epidermes of intact Vicia leaves are not more sensitive to light than those in adaxial epidermes, and that stomata in leaves of this plant do not respond to light alone. Additional factors in bulk leaf tissue probably produce coordinated stomatal opening on upper and lower leaf epidermes to optimally meet photosynthetic requirements of the whole leaf for CO₂.     

Effect of human chorionic gonadotrophin on 7,12-dimethylbenz[a]anthracene-induced DNA binding and repair synthesis by rat mammary epithelial cells

The administration of the placental hormone human chorionic gonadotrophin (HCG) to 50-day-old virgin Sprague--Dawley rats has been shown to reduce the incidence of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary cancer. We now report from studies using rat mammary epithelial cells in culture that the anti-carcinogenic effect of HCG may be related to its effect on DNA binding of DMBA and on DNA repair. The results showed that the level of excision repair in cells derived from young virgin (YV) rats grown in the presence of HCG (10 units/ml) was 2.5-4.0 times higher than the level exhibited by control YV cells and 1.5-2.5 times over that obtained for cells from old virgin and parous rats. The effect of HCG on DMBA-DNA binding was also determined in YV cells cultured in the presence of HCG (10 units/ml). Results from this study indicated that DMBA-DNA binding was inhibited by 30-40% in HCG-treated cells as compared to control cells. DNA binding of DMBA was also determined with mammary epithelial cells from YV rats which were given subcutaneous injections of HCG (5 units/rat) 5 times per week for 4 weeks. Using this in vivo-in vitro protocol, DMBA-DNA binding was 17-51% lower in cells from HCG-treated rats than in cells derived from control saline-treated rats. These results suggest that the protective effect provided by HCG against DMBA-induced mammary tumorigenesis may be attributed to its ability to inhibit binding of the carcinogen to mammary cell DNA and to its ability to increase the level of excision repair in the cells.