Localization and cloning of Xp21 deletion breakpoints involved in muscular dystrophy

Summary Twenty-nine deletion breakpoints were mapped in 220 kb of the DXS164 locus relative to potential exons of the Duchenne and Becker muscular dystrophy gene. Four deletion junction fragments were isolated to acquire outlying Xp21 loci on both the terminal and centromere side of the DXS164 locus. The junction loci were used for chromosome walking, searches for DNA polymorphisms, and mapping against deletion and translocation breakpoints. Forty-four unrelated deletions were analyzed using the junction loci as hybridization probes to map the endpoints between cloned Xp21 loci. DNA polymorphisms from the DXS164 and junction loci were used to follow the segregation of a mutation in a family that represents a recombinant. Both the physical and genetic data point to a very large size for this X-linked muscular dystrophy locus.     

Reversible cellular senescence: implications for immortalization of normal human diploid fibroblasts.

IMR-90 normal human diploid fibroblasts, transfected with a steroid inducible mouse mammary tumor virus-driven simian virus 40 T antigen, were carried through crisis to yield an immortal cell line. Growth was dependent on the presence of the inducer (dexamethasone) during both the extended precrisis life span of the cells and after immortalization. After dexamethasone removal, immortal cells divided once or twice and then accumulated in G1. These results are best explained by a two-stage model for cellular senescence. Mortality stage 1 (M1) causes a loss of mitogen responsiveness and arrest near the G1/S interface and can be bypassed or overcome by the cellular DNA synthesis-stimulating activity of T antigen. Mortality stage 2 (M2) is an independent mechanism that is responsible for the failure of cell division during crisis. The inactivation of M2 is a rare event, probably of mutational origin in human cells, independent of or only indirectly related to the expression of T antigen. Under this hypothesis, T-antigen-immortalized cells contain an active but bypassed M1 mechanism and an inactivated M2 mechanism. These cells are dependent on the continued expression of T antigen for the maintenance of immortality for the same reason that precrisis cells are dependent on T antigen for growth: both contain an active M1 mechanism.     

5,6,7,8-Tetrahydromethanopterin-dependent enzymes involved in methanogenesis

Abstract In the process of methanogenesis, 5,6,7,8-tetrahydromethanopterin (H₄MPT) is the carrier of the C₁ unit at the formyl through methyl state of reduction. By the transfer of a formyl group from formylmethanofuran, 5-formyl- and 10-formyl-H₄MPT are formed in hydrogenotrophic and methylotrophic organisms, respectively. Cyclohydrolysis of the 5- and 10-formyl derivatives then yields 5,10-methenyl-H₄MPT, which is reduced in two subsequent coenzyme F₄₂₀-dependent reactions to 5-methyl-H₄MPT. Following the transfer of the methyl group to coenzyme M, the substrate of the terminal step in methanogenesis, methylcoenzyme M, is produced. In this paper properties of the enzymes catalyzing the individual H₄MPT-dependent reactions are discussed.     

Transformation with a mutant Arabidopsis acetolactate synthase gene renders tobacco resistant to sulfonylurea herbicides

Summary A gene encoding acetolactate synthase was cloned from a chlorsulfuron-resistant mutant of Arabidopsis. The DNA sequence of the mutant gene differed from that of the wild type by a single base pair substitution. When introduced into tobacco by Ti plasmid-mediated transformation the gene conferred a high level of herbicide resistance. These results suggest that the cloned gene may confer agronomically useful levels of herbicide resistnace in other crop species, and that it may be useful as a selectable marker for plant transformation experiments.     

Correction of chlorophyll-defective male-sterile winter oilseed rape (Brassica napus) through organelle exchange: Characterization of the chlorophyll deficiency

Jarl, C. I., Ljungberg, U. K. and Bornman, C. H. 1988. Correction of chlorophyll-defective male-sterile winter oilseed rape ( Brassica napus ) through organelle exchange: Characterization of the chlorophyll deficiency. - Physiol. Plant. 72: 505–510.
As is known, the introduction of male-sterile Raphanus sativus L. cytoplasm into Brassica napus L. results in male-sterile oilseed rape plants, which display a temperature-related chlorophyll defect. The influences of temperature and irradiance on this defect were investigated. Compared to a line of normal (green phenotype) male-fertile oilseed rape, the male-sterile line had reduced chlorophyll content, fewer chloroplasts per cell, an altered ultrastructure of the chloroplasts and reduced activities of both photosystems, although the relative amounts of the photosystems and the chlorophyll a/b ratio were similar. The lower activity of the photosystems is explained by a decreased functional antennae size and a reduced efficiency in the interactions between the nuclear-encoded light-harvesting proteins and the reaction centres coded for by the plastome. Some thylakoid polypeptides differed in proportion between the male-fertile line with green phenotype and the male-sterile line with chlorotic phenotype. Characters, in which the two lines exhibited differences, are ascribed to difficulties in molecular communication between the oilseed rape nucleus and the radish cytoplasm, which are combined in the deficient male-sterile line.     

Constitutive and transport-related endocytotic pathways in turtle bladder epithelium

Summary Proton secretion in the urinary bladder of the fresh-water turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo-and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15°C. In addition, serosal application of 0.5mm acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15°C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1mm serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.     

Lymphokine production by human melanoma tumor-infiltrating lymphocytes

Summary Lymphokine production by human melanoma tumor-infiltrating lymphocytes (TIL) was studied. Uncultured TIL produced interferon (IFN), but not interleukin-2 (IL-2) or IL-4, in response to anti-CD3 mAb or IL-2. In bulk cultures, IL-2-activated TIL displaying autologous tumor-specific cytotoxicity (CTL-TIL) produced IFN in culture with medium alone, whereas IL-2-activated noncytotoxic TIL did not. Addition of anti-CD3 mAb or autologous tumor cells up-regulated IFN production in IL-2-activated TIL from 10 of 12 or 6 of 12 cases respectively. Those from 4 of 12 cases (2 CTL-TIL and 2 noncytotoxic TIL) produced IL-2 in culture with medium alone. At the clonal level, 5 (4 CD4⁺ and 1 CD8⁺) of 7 autologous tumor-specific CTL clones derived from TIL and 3 (2 CD4⁺ and 1 CD8⁺) of 7 noncytotoxic TIL clones produced IFN in culture with medium alone, which was up-regulated by adding anti-CD3 mAb. Two IFN-producing CTL clones tested produced IL-2 in 4 ×-concentrated supernatants from a 3.5-h culture with medium alone. Furthermore, 2 IFN-producing CTL clones tested expressed mRNA for both IFN and IL-2. IL-2 production and its mRNA expression were up- or down-regulated, respectively, by adding anti-CD3 mAb or autologous tumor cells. IL-4 production was not observed in culture either with medium alone or with IL-2 in any of the cells described above. Anti-CD3 mAb was required for IL-4 production in 3 of 12 IL-2-activated TIL, 2 of 6 CTL clones, and none of 5 noncytotoxic TIL clones. In summary, IFN production was characteristic of melanoma TIL. Some autologous tumor-specific CTL in TIL are suggested to be productive of IL-2 and IFN under unstimulated conditions, both being required for self-activation in an autocrine loop.This work was supported in part by grant CA-47891 from the National Cancer Institute     

Measurement and effects of gel matric potential and expressibility on production of morphogenic callus by cultured sugarbeet leaf discs

During studies to optimize production of morphogenic callus from cultured leaf discs of sugarbeet (Beta vulgaris L.) large differences were observed associated with the gelling agent employed. Water availability, as determined mainly by gel matric potential, was found to be the dominant factor. A simple method was devised to measure the relative matric potential of different gels. A precisely moistened filter-paper disc was placed on the gel surface, allowed to equilibrate, removed and weighed. The relative gain or loss of water from the paper disc was a measure of the matric potential of the gel and varied with both gel type and concentration. Leaf disc expansion and production of callus-derived embryos and shoots were shown to be directly proportional to gel matric potential. Water availability may also be affected by the ease with which liquid is expressed from gels in response to localized pressure caused by explant expansion and contortion. This property, called gel expressibility, was easily measured with a weight and capillary pipette and shown also to vary with gel type and concentration. Validity of the technique for measuring relative matric potential was verified physiologically by culturing leaf discs on filter-paper overlays to eliminate expressibility differences among gels. Additionally, comparison of leaf disc growth on uncovered gel surfaces versus filter-paper overlays demonstrated the contribution of liquid expression to overall water availability. Expression of liquid by explants on uncovered gel surfaces greatly enhanced the production of morphogenic callus.     

Lymphocyte activation and serine-esterase induction following recombinant interleukin-2 infusion for lymphomas and acute leukaemias

Summary C57BL mice inoculated with radiation leukemia virus (RadLV) develop preleukemic cells long before the onset of leukemia. These cells are potentially immunogenic but fail to elicit an immune response in the host because of the appearance of virus-specific suppressor T cells. We have studied the effect of polysaccharide K (PSK) on the generation of RadLV-specific cell-mediated immune responses in vitro. Long-term exposure to PSK in culture potentiated the ability of immunized T cells to respond to a RadLV-induced lymphoma. It also abrogated the suppressive activity of suppressor T cells and simultaneously boosted the ability of reactive T cells to respond. The dual immunostimulating activity of PSK resulted in the generation of T cytotoxic lymphocytes that could lyse lymphoma cells in vitro. The results suggest that PSK could be used as a prophylactic immune response modifier in preleukemia.