Postsynaptic localization of the alpha receptor-mediated stimulation of phosphatidylinositol turnover in pineal gland.

The phosphatidylinositol effect in rat pineal gland (defined as the enhanced incorporation of P_i into phosphatidylinositol which is elicited by agonists) is mediated through alpha-adrenergic receptors. Experiments with glands from animals treated with 6-hydroxydopamine, with dispersed pinealocytes and with a number of relatively specific pre- and postsynaptic alpha-adrenergic agonists and antagonists have shown the receptors involved to be located at postsynaptic sites.     

Lectin-mediated induction of IL-4-producing CD4+ T cells.

Cultured murine CD4+ T cells have been shown to differentiate into IL-2 or IL-4-producing subsets. The factors responsible for the development of CD4+ T cells which produce IL-2 but not IL-4 and cells capable of producing IL-4 but not IL-2 are unknown. Here we describe a system that allows the controlled induction of IL-2- or IL-4-producing T cells after one single round of activation. Freshly isolated CD8-depleted T cells were activated with various polyclonal T cell activators for 48 h, washed, and then expanded under different conditions. IL-2 and IL-4 production were induced by restimulation of T cells and were measured with CTLL cells that respond to both cytokines and mAb to IL-2 and IL-4. T cells produced mainly IL-2 and small amounts of IL-4 when restimulated after expansion culture for 12 days with rIL-2 alone. However, after expansion for 12 days in the presence of rIL-2 plus Con A, we observed a 30- to 100-fold up-regulation of IL-4 activity and a 100-fold down-regulation of IL-2 when assessed by responses of CTLL cells incubated with the supernatant of restimulated T cells and by responses of CTLL cells cocultured with restimulated cells. An increase of IL-4 and decrease of IL-2 was also observed when the results were based on the cell numbers at the beginning of the expansion culture. The induction of IL-4 and the down-regulation of IL-2 1) were not reproduced with alpha-methyl-mannoside-treated supernatant of Con A-stimulated spleen cells, 2) were not dependent on the presence of large numbers of APC, 3) did not result from differential consumption of lymphokines after restimulation, 4) were not due to a difference in the time course of IL-2 or IL-4 release in either T cell population, and 5) were obtained regardless of the agents used to activate or to restimulate the T cells. Because Con A remained detectable on the T cell surface and because expansion of activated T cells with IL-2 plus Con A for several days was necessary, our results indicate that mainly IL-4-producing CD4+ T cells can be induced by prolonged engagement of T cell surface molecules.     

Effect of inorganic cations on phase transitions.

The effect of protons and cations on the crystal (gel)-to-liquid crystal transition temperature Tm of isoelectric and negatively charged phospholipids are summarized. The general trends emerging are as follows: Tm depends on the state of ionization of the phospholipid in that Tm-vs-pH-curves parallel the titration curve of the phospholipid. Protonation of phospholipids causes Tm to increase, deprotonation or ionization has the opposite effect. The effects of cations on the Tm of phospholipids may be grouped into non-specific and specific effects. Unspecific effects of cations such as the screening of negative charges of the phospholipid polar group are qualitatively similar to protonation: Tm increases, in the order monovalent less than divalent less than trivalent cations and the effects on negatively charged phospholipids are larger than those on isoelectric phospholipids. Unspecific, electrostatic effects on Tm are reasonably well accounted for by the Gouy-Chapman theory. If, however, specific binding comes into play and/or electrostatic effects are accompanied by changes in phospholipid structure, simple, electrostatic theories fail to explain the observed changes in Tm. The crystal (gel)-to-liquid crystal transition is also a function of the degree of hydration: Tm generally decreases with increasing hydration reaching a plateau in excess H2O. In addition to screening of electric charges, ions may exert yet another non-specific effect: ions may affect Tm indirectly by competing with the phospholipid polar group for water of hydration. This indirect effect plays a role at high ionic strength and/or at low hydration of the phospholipid. Specific binding of cations to negatively charged phospholipids can lead to tight associations of the metal ion with the lipid polar group. Isothermal crystallization of the phospholipid bilayer is induced that is accompanied by a total or partial loss of water of hydration resulting in a marked increase in Tm. For instance, in crystalline Ca2(+)-phosphatidylserine complexes Tm is increased by more than 100 degrees C.     

Membrane proteins exposed on the external side of the intestinal brush-border membrane have fusogenic properties

The intestinal brush-border membrane contains one or several membrane proteins that mediate fusion and/or aggregation of small unilamellar egg phosphatidylcholine vesicles. The fusion is accompanied by a partial loss of vesicle contents. Proteolytic treatment of the brush-border membrane with proteinase K abolishes the fusogenic property. This finding suggests that the fusogenic activity is associated with a membrane protein exposed on the external or luminal side of the brush-border membrane. Activation of intrinsic proteinases of the brush-border membrane liberates water-soluble proteins (supernate proteins). These proteins behave in an analogous way to intact brush-border membrane vesicles; they induce fusion of egg phosphatidylcholine vesicles and render the egg phosphatidylcholine bilayer permeable to ions and small molecules (Mr less than or equal to 5000). Furthermore, supernate proteins mediate phosphatidylcholine and cholesterol exchange between two populations of small, unilamellar phospholipid vesicles. Supernate proteins are fractionated on Sephadex G-75 SF yielding three protein peaks of apparent Mr greater than or equal to 70,000, Mr = 22,000 and Mr = 11,500. All three protein fractions show similar phosphatidylcholine-exchange activity, but they differ in their effects on the stability of egg phosphatidylcholine vesicles. The protein fraction with an apparent Mr greater than or equal to 70,000 has the highest fusogenic activity while the protein fraction of apparent Mr = 11,500 appears to be most effective in rendering the egg phosphatidylcholine bilayer permeable.     

Central role for TCR/CD3 ligation in the differentiation of CD4+ T cells toward A Th1 or Th2 functional phenotype.

Activated CD4+ T cells can be classified into distinct subsets; the most divergent among them may be considered to be the IL-2 and IFN-gamma-producing Th1 clones and the IL-4 and IL-5-producing Th2 clones. Because Th1 and Th2 clones can usually be detected only after several months of culture, we used conditions that modulate the IL-2 and IL-4 production in short term culture. Here we show that freshly isolated and subsequently in vitro-activated CD4+ T cells that were cultured for 11 days with rIL-2 and restimulated showed a IFN-gamma+ IL-2+ IL-3+ IL-4- IL-5- pattern. Because these cells were not capable of providing B cell help for IgG1, IgG2a, or IgE in an APC- and TCR-dependent T-B cell assay, they expressed a phenotype typical for most Th1 clones. In contrast, activated T cells that were cultured for 11 days with IL-2 plus a mAb to CD3 and then restimulated produced a IFN-gamma- IL-2- IL-3+ IL-4+ IL-5+ pattern. These cells were capable of providing B cell help for IgG1, IgG2a, and IgE synthesis and thus presented a phenotype typical for Th2 clones. Similar results were observed when mitogenic mAb to Thy-1.2 or to framework determinants of the alpha beta TCR were used. The induction of Th1- and Th2-like cells did not depend on the relative expression of CD44 or CD45 by the T cells before activation in vitro. Because the incubation of activated T cells with anti-CD3/TCR mAb induced high unrestricted lymphokine production, the latter might be responsible for the Th2-like lymphokine pattern observed after restimulation. To address this point, TCR V beta 8+ and V beta 8- T cell blasts were co-cultured in the presence of mAb to V beta 8. After restimulation, V beta 8+ cells had a IL-4high IL-2low phenotype and V beta 8- cells had a IL-4low IL-2high phenotype. This demonstrates that TCR ligation but not lymphokines alone are capable of inducing Th2-like cells, and this points out a central role for the TCR in the generation of T cell subsets.     

Methane oxidation by methanotrophs and its effects on the phosphate flux over the sediment-water interface in a eutrophic lake

The effect of methane oxidation in aerobic sediment on oxygen consumption and phosphate flux was investigated in diffusion chambers. The diffusion chambers consisted of two compartments separated by a Teflon membrane. In the upper chamber a thin sediment layer was present and the lower chamber was continuously flushed with gas. The hydrophobic membrane allowed for diffusion of gases from the lower chamber through the sediment layer toward the headspace of the upper chamber. In experiments with a methane oxidation rate of 9.8 mmol m^–2 day^–1, the oxygen consumption rate increased by a factor of two compared with controls without methane oxidation (8.6 vs 17.7 mmol m^–2 day^–1). Methane oxidation significantly decreased oxygen penetration depth (2.5–4.0 vs 1.0–2.0 mm). However, despite the shrinkage of the oxidized microlayer, no differences were found in phosphate flux across the sediment water interface. Batch experiments with standard additions of methane revealed that the growth of methanotrophic bacteria contributes to the phosphate uptake of aerobic sediment. From the batch experiments a molar ratio of carbon to phosphate of 45 mol:mol was calculated for the growth of methanotrophs. Results suggest that a decrease in chemical phosphate adsorption caused by a decrease in the oxygen penetration depth could be compensated for entirely by the growth of methanotrophic bacteria. Send offprint requests to: A.J.C. Sinke     

Precursor-product relationship of larger to smaller molecular forms of the BAL 31 nuclease from Alteromonas espejiana: preferential removal of duplex exonuclease relative to endonuclease activity by proteolysis

Two molecularly and kinetically distinct major species of the extracellular nuclease BAL 31 from Alteromonas espejiana, previously characterized as the "fast" (F) and "slow" (S) BAL 31 nucleases, have been evidenced to derive from proteolysis starting from a still larger (approximately 120 kDa) precursor nuclease. The expected protease activity in the culture fluid has been confirmed and is strongly dependent on the cell growth phase. The disappearance of the largest nuclease species with the concomitant sequential appearance of first the F and then the S species has been demonstrated for nuclease obtained from culture supernatants as a function of cell growth phase. Nuclease from periplasmic extracts displayed very little of the F and S nucleases. Treatment of purified F nuclease with Pronase or subtilisin readily converted it to species with only a few percent of the native exonuclease activity against duplex DNA but retaining much of the initial activity against single-stranded DNA. Electrophoresis in nuclease-detecting gels demonstrated a parallel conversion of the larger species to one indistinguishable in molecular weight from the S species. The observed loss of exonuclease activity could correspond to the conversion of the F to the S nuclease. However, treatment of S nuclease with subtilisin resulted in a drastic reduction of exonuclease activity of this enzyme on duplex DNA with retention of most of the activity against single-stranded and nicked circular duplex DNA substrates. Evidence of internal proteolysis of the S nuclease could be seen after electrophoresis in denaturing gels but only after the denaturation buffer was adjusted to 6 M in urea. The preferential removal of the exonuclease activity may enhance the usefulness of the BAL 31 nuclease in such applications as heteroduplex mapping.     

A locus for familial hypertrophic cardiomyopathy is closely linked to the cardiac myosin heavy chain genes, CRI-L436, and CRI-L329 on chromosome 14 at q11-q12

We report that a gene responsible for familial hypertrophic cardiomyopathy (HC) is closely linked to the cardiac alpha and beta myosin heavy chain (MHC) genes on chromosome 14q11. We have recently shown that probe CRI-L436, derived from the anonymous DNA locus D14S26, detects a polymorphic restriction fragment that segregates with familial HC in affected members of a large Canadian family. Using chromosomal in situ hybridization, we have mapped CRI-L436 to chromosome 14 at q11-q12. Because the cardiac MHC genes also map to this chromosomal band, we have determined the genetic distances between the cardiac beta MHC gene, D14S26, and the familial HC locus. Data presented here show that these three loci are linked within 5 centimorgans on chromosome 14 at q11-q12. The possibility that defects in either the cardiac alpha or beta MHC genes are responsible for familial HC is discussed.     

Gene shuttling: moving of cloned DNA into and out of eukaryotic cells.

Successful shuttling of cloned DNA in eukaryotic cells should allow isolation of expressed genes. We tested the utility of cosmids for moving DNA into and out of eukaryotic cells. The unique cleavage of DNA at the cos site by the terminase function of lambda was exploited to maintain the linkage between the vector and inserted gene sequences, a prerequisite for successful rescue of the transforming DNA from high molecular weight DNA of the eukaryotic transformant. A cosmid recombinant containing the HSV thymidine kinase gene and a lambda recombinant containing the chicken thymidine kinase gene were used to test the feasability of this method. It was found that these recombinants can be rescued with high efficiency from DNA of HAT-resistant cells.