Glucose metabolism and internal pH of Lactococcus lactis subsp. lactis cells utilizing NMR spectroscopy

The metabolism of glucose was studied in Lactococcus lactis subsp. lactis CNRZ 125 by ¹³C NMR. The initial rate of glucose utilization was higher for exponential phase cells than for stationary phase cells [150 vs 85 nmol g (dry wt)^-1 s^-1]. ³¹P NMR was used to determine changes in glycolytic phosphorylated intermediates (fructose-1,6-diphosphate, dihydroxyacetone phosphate and phosphoglycerate). The internal pHs of L. lactis subsp. lactis CNRZ 141 and CNRZ 125 were also measured by ³¹P NMR as a function of the external pH during growth. When the external pH was 6·8, the internal pHs of strain CNRZ 141 and CNRZ 125 were similar, 7·4. After the external pH had decreased to 5·5, the internal pH of strain CNRZ 141 had declined by 0·6 unit, whereas that of strain CNRZ 125 had decreased by only 0·2 unit of pH.     

RelA/p65 is a molecular target for the immunosuppressive action of protein kinase A.

Stimulation of the protein kinase A (PKA) signalling pathway exerts an inhibitory effect on the proliferation of numerous cells, including T lymphocytes. In CD4+ T helper cells, stimulation of PKA leads to suppression of interleukin 2 (IL-2) induction, while induction of the genes coding for the lymphokines IL-4 and IL-5 is enhanced. We show that the differential effect of PKA activity on induction of the IL-2 and IL-4 genes is mediated through their promoters. One major target of the suppressive effect of PKA is the kappa B site in the IL-2 promoter. A kappa B site is missing in the IL-4 promoter. Mutations preventing factor binding to the IL-2 kappa B site result in a loss of PKA-mediated suppression of IL-2 promoter activity. Furthermore, activation of the PKA signalling pathway impairs the inducible activity of multiple kappa B sites of the IL-2 promoter, but not of other factor binding sites. The reduction in activity of kappa B sites in activated and PKA-stimulated T cells is accompanied by changes in the concentration and DNA binding of Rel/NF-kappa B factors. Stimulation of the PKA pathway in Jurkat T cells with the PKA activator forskolin leads to an increase in synthesis of c-Rel and p105/p50, while synthesis of p65/RelA remains unchanged. However, nuclear translocation and DNA binding of p65 is distinctly impaired, probably due to a retarded degradation of I kappa B-alpha. In a similar way, stimulation of the PKA signalling pathway inhibits nuclear translocation of p65 and generation of nuclear kappa B complexes in peripheral T lymphocytes from murine lymph nodes. These results indicate that PKA-mediated suppression of NF-kappa B activity plays an important role in the control of activation of peripheral T lymphocytes.     

Phenotypic features of breast cancer cells overexpressing ornithine-decarboxylase

Polyamines (PA) have been shown to be critical mediators of estradiol-induced breast cancer cell proliferation. This finding suggests that constitutive activation of the PA pathway may promote tumor progression, possibly leading to hormone independence. To test this hypothesis, we transfected hormone-responsive MCF-7 breast cancer cells with a complementary DNA coding for ornithine-decarboxylase (ODC), the first rate-limiting enzyme in PA biosynthesis. Marked ODC over-expression observed in stably transfected clones was associated with a selective increase in cellular putrescine content, while spermidine and spermine levels were not altered. ODC-overexpressing MCF-7 cells were resistant to the antiproliferative effects of low but not high concentrations of the enzyme inhibitor, α-difluoromethylornithine. In agreement with our hypothesis, sensitivity to the growth-promoting action of estradiol was reduced by approximately one third (P < 0.001) in ODC-overexpressing MCF-7 cells compared with vector-only transfected clones. Basal growth under anchorage-dependent conditions was only marginally increased by ODC overexpression (P = 0.048), while clonogenicity in soft agar was actually reduced. These data suggest that activation of PA biosynthesis may contribute in part to the acquisition of estrogen independence by breast cancer cells. Since only putrescine content was increased as a result of ODC overexpression, these data may underestimate the overall influence of the PA pathway on breast cancer phenotype. © 1995 Wiley-Liss, Inc.     

Functional characterization of lymphoid cells generated in serum-deprived culture stimulated with stem cell factor and interleukin 7 from normal and autoimmune mice

We have investigated the phenotypic and functional characteristics of murine pre-B cells obtained in semisolid and liquid culture with stem cell factor (SCF) and interleukin 7 (IL-7). Both serum-supplemented and serum-deprived culture conditions were used. The source of bone marrow cells was either normal mice (CD1 and C3H) or the lupus strain of mice MRL/Ipr and its congenic strain MRL/+. SCF (100 ng/ml) and IL-7 (250 ng/ml) supported murine B cell proliferation in vitro from all the murine strains analyzed both in serum-supplemented and serum-deprived conditions. Maximal colony growth was observed in both cases when the factors were used in combination. The growth factors alone induced some colony growth in serum-supplemented cultures but were either ineffective or had modest activity in serum-deprived cultures. Cells harvested from the colonies or generated in liquid cultures and stimulated with SCF + IL-7 in the absence of serum had almost exclusively a pre-B cell phenotype (BP-1+, B220+, slg-, CD4-, CD8-, Mac-1, RB-6-). Both the maximal colony growth in semisolid culture and the maximal number of cells in liquid culture were observed at day 12–14. At this time, the pre-B cells failed to differentiate further and started to die. Pre-B cells generated in vitro were, however, capable of differentiating in vivo. SCID mice injected with 2 × 10⁶ pre-B cells had readily detectable serum levels of IgM (54 ± 26 m?g/ml) and IgG (60 ± 95 m?g/ml) at 4 weeks and 6 weeks posttransplantation, respectively. Mature B and T cells of the donor major histocompatibility complex type were detected in the SCID mice at sacrifice 14 weeks posttransplantation. These data indicate that purified (>80% BP-1+) populations of functional pre-B cells can be grown from murine bone marrow of normal mice as well as of lupus mice in serum-deprived cultures stimulated with SCF and IL-7. These cultures, therefore, provide a highly enriched source of pre-B cells but also contain T cell precursors that differentiate upon adoptive transfer into SCID mice. © 1995 Wiley-Liss, Inc.     

Specific induction of self-discrimination by follicle cells in Ciona intestinalis oocytes

Self-incompatibility, a mechanism that prevents self-fertilization in ascidians, is based on the ability of the oocyte vitelline coat to distinguish and accept only heterologous spermatozoa. In Ciona intestinalis self-discrimination is established during late oogenesis and is contributed or controlled by products of the overlying follicle cells. In this study we have further investigated the role of the follicle cells in the onset of self-discrimination by using in vitro maturation of ovarian oocytes deprived of the follicle cells and incubated with either autologous or heterologous follicle cells. Fertilization assays demonstrate that the action of the follicle cells is exerted even when they are detached from the vitelline coat and that only autologous follicle cells can promote the induction of self-sterility on the egg coat. Electron microscopy of the oocytes during maturation reveals that the switch from self-fertility to self-sterility is accompanied by the appearance of a thin electron-dense layer on the outer surface of the vitelline coat. We suggest that the formation of this layer is the result of the interaction between products of the follicle cells and the autologous vitelline coat.     

Long-Term Induction of Tyrosine Hydroxylase Expression: Compensatory Response to Partial Degeneration of the Dopaminergic Nigrostriatal System in the Rat Brain

Abstract: The present study was undertaken to examine the adaptive changes occurring 1 and 6 months after moderate or severe unilateral 6-hydroxydopamine-induced lesions confined to the lateral part of the rat substantia nigra pars compacta (SNC). The expression of tyrosine hydroxylase (TH) enzyme was analyzed in the remaining dopaminergic nigral cell bodies and in the corresponding striatal nerve endings. In the cell bodies of the lesioned SNC, TH mRNA content was increased (+20 to +30%) 6 months after the lesion without changes in cellular TH protein amounts. The depletion of TH protein in the nerve terminal area was less severe than the percentage of cell loss observed in the SNC at 1- and 6-month postlesion intervals. Moreover, the decrease in TH protein in the ipsilateral striatum was less pronounced 6 months after lesion than 1 month after. That no corresponding change in TH protein content was observed in the cell bodies at a time when TH increased in nerve terminals suggests that the newly synthesized protein is probably rapidly transported to the striatal fibers. These results suggest the existence of a sequence of changes in TH expression between cell bodies and fibers, occurring spontaneously after partial denervation of the nigrostriatal pathway.     

RNA aptamers specifically interact with the prion protein PrP.

We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein rPrP23-231 (rPrPc) fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST::rPrP90-231 (rPrP27-30), which lacks 67 amino acids from the PrP N terminus. The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition. Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice (C57BL/6), hamsters (Syrian golden), and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice (prn-p(0/0)). Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize PrP27-30 in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.     

Nuclear import and export of influenza virus nucleoprotein.

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.