Endogenous gibberellin A1 levels control thermoperiodic stem elongation in Pisum sativum

Gibberellin (GA) is believed to be involved in thermoperiodic stem elongation. With this in mind, we studied the correlation between gibberellin A₁ (GA₁) levels and stem elongation affected by alternating day (DT) and night temperature (NT) in 5 genotypes of Pisum sativum differing in their degree of dwarfism. The endogenous GA content in the tissue of two of the genotypes was determined by combined gas chromatography and mass spectrometry. The wild genotype developed 40 to 50% shorter stems and internodes under a low DT and high NT combination (negative difference [DIF] between DT and NT, DT/NT 15.5/21.5 or 14/24°C) than under the opposite regime of high DT and low NT (positive DIF, DT/NT 22.5/16.5 or 24/14°C). The GA biosynthetic mutants ls and le, and the auxin and brassinosteroid mutant lkb responded in a similar way, but not as strongly as the wild type. The stem length of the GA-insensitive slender mutant (la cry^s) was reduced by only 8% under negative compared to positive DIF. In the wild type endogenous GA levels decreased by 60% from positive to negative DIF in the upper part of the stem. Further, there was a corresponding decrease in the levels of precursors to GA₁, i.e. GA₅₃, GA₄₄, GA₁₉ and GA₂₀, while 2β-hydroxylated GA₂₀ and GA₁, GA₂₉ and GA₈, respectively, were unaffected by DIF. A similar increase in the ratios of GA₂₉ to GA₂₀ and GA₈ to GA₁ from positive to negative DIF was seen in the stem tissue of the le mutant as in the wild type. The temperature regimes affected the levels of GA₁ and its precursors in combined leaf and petiole samples and in the shoot tip in a similar manner as in the stem tissue. However, the different temperature regimes did not affect the ratio of GA₈/GA₁ in the shoot tip. The results indicate that altered stem elongation of the pea plants in response to diurnal temperature alternations may be mediated by changes in endogenous levels of GA₁. The GA₁ levels may be controlled by an effect of DIF on both biosynthetic and inactivation steps.     

Shape and fine structure of nucleoids observed on sections of ultrarapidly frozen and cryosubstituted bacteria.

Very rapidly frozen cells of Escherichia coli and Bacillus subtilis were substituted at low temperature into acetone with 1% OsO4 and embedded in Epon. They showed ribosome-free spaces filled with globular and fibrillar material of up to 15 nm. The sizes of structures seen do not exclude DNA superstructures such as supercoils, aggregates, and nucleosomes. With the Feulgen analog osmium-ammines stain, DNA was localized within the ribosome-free space. The bulk of DNA, the nucleoid, is therefore a major part of, or identical to, the main ribosome-free space. The ribosome-free space would correspond directly to the light microscopy phase-contrast image of nucleoids in living bacteria. The shape of the ribosome-free space does not reflect intracellular salt concentrations, nor do the Feulgen-positive areas. The previously observed dependency on the salt concentration of the growth medium seems to be due to permeabilization induced by the chemical fixative at room temperature. The ribosome-free space is more cleft in appearance than the nucleoid obtained by fixation with OsO4 but more confined than its very dispersed form found after aldehyde fixation.     

Productive phage infection in Escherichia coli with reduced internal levels of the major cations.

Bacteriophage-induced changes in the intracellular levels of the major cations of Escherichia coli were studied to investigate the role of ion concentrations for bacteriophage assembly in vivo. Infection of E. coli by phage T4, P1, or lambda caused a transient reduction of intracellular levels of potassium, magnesium, and polyamines. Phages T3 and T7, however, had no detectable effect on the cation concentrations within the cell. In all cases, any reduction in the ion concentrations was restored later in infection. When the intracellular potassium concentration was lowered from 325 to 150 mM with a different osmotic growth medium, the number of phage progeny was only slightly reduced (by a factor of two). On additional reduction of the intracellular magnesium concentration from 100 to 50 mM by adding the antibiotic polymyxin B to the infected cells, T4 infections, but not T3 or T7, were markedly affected. These studies show that T3, T4, and T7 phage assembly can efficiently occur in vivo over a broad spectrum of ion concentrations.     

Function of the alpha 1 beta 2 gamma 2S gamma-aminobutyric acid type A receptor is modulated by protein kinase C via multiple phosphorylation sites.

Activation of protein kinase C (PKC) results in down-modulation of the gamma-aminobutyric acid type A (GABAA) receptor. In this study, the recombinant subunit combination alpha 1 beta 2 gamma 2S was expressed in Xenopus oocytes. The resulting channel was shown to be modulated by 2 microM oleoylacetylglycerol or, stereo-specifically, by low concentrations (10 nM) of the phorbol ester 4 beta-phorbol 12-myristate 13-acetate. By site-specific mutagenesis, we altered the serine or threonine residues of consensus phosphorylation sites for PKC in the large, intracellular domain of alpha 1, beta 2, and gamma 2S. Mutant subunits were co-expressed with wild type subunits to yield alpha 1 beta 2 gamma 2S combinations. All of the tested 14 mutations did not affect the level of expression of GABA current. Two of these mutations, Ser-410 in beta 2 and Ser-327 in gamma 2S, resulted in a significant reduction of the effect of the activator of PKC, 4 beta-phorbol 12-myristate 13-acetate, on the GABA current amplitude. Thus, we have identified two single serine residues, Ser-410 in the subunit beta 2 and Ser-327 in gamma 2S, as phosphorylation sites of a PKC endogenous to Xenopus oocytes. Co-expression of the mutant subunits suggests that phosphorylation of both sites is required for a full, PKC-mediated down-regulation of GABA currents.     

Point mutations affecting antagonist affinity and agonist dependent gating of GABAA receptor channels.

Two variant amino acid sequences, which differ in a single amino acid residue, have been reported for the alpha 1-subunit of the rat brain GABAA receptor. We separately co-expressed these two variants in Xenopus oocytes, in combination with beta 2 and gamma 2. This experiment showed that substitution of alpha 1-Phe64 by Leu strongly decreases the apparent affinity for GABA dependent channel gating from 6 microM to 1260 microM. Starting from this observation, we used in vitro mutagenesis to obtain information relevant for the localization of the agonist/antagonist binding site in the GABAA receptor. Homologous mutation in alpha 5 had similar consequences for alpha 5 beta 2 gamma 2. Homologous mutation in beta 2 and gamma 2 resulted in intermediate and small shifts in EC50, respectively. The apparent affinities of the competitive antagonists bicuculline methiodide and SR95531, the latter sharing close structural similarity with the agonist GABA, were decreased 60- to 200-fold by these mutations in alpha-subunits. Interestingly, these affinities remained nearly unaffected upon introduction of the homologous mutations in beta 2 and gamma 2, or upon mutation of the neighbouring amino acid in alpha 1, Phe65 to Leu. These results suggest close functional and structural association of alpha-subunits with the agonist/antagonist binding site, and involvement of N-terminal portions of the extracellular domains of all subunits in the gating of the channel.     

Use of on-section immunolabeling and cryosubstitution for studies of bacterial DNA distribution.

Escherichia coli cells were very rapidly frozen and substituted at a low temperature with 3% glutaraldehyde in acetone. Infiltration and embedding with Lowicryl K4M were carried out at -35 degrees C. This procedure resulted in good structural preservation of both the nucleoid morphology and its DNA plasm, such that immunolabeling with the protein-A gold technique could be carried out. With antibodies specific for either double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA), it was shown that dsDNA was present throughout the nucleoid but that ssDNA was located on the nucleoid periphery. Chloramphenicol-treated cells, in which protein synthesis but not DNA replication is stopped, produced a characteristic ringlike nucleoid shape and had both dsDNA and ssDNA present throughout the annular section of the DNA plasm. The relationship between metabolically active DNA and overall bacterial genome organization is discussed.     

Assembly and disassembly of bacteriophage T4 polyheads

The assembly of the product of bacteriophage T4 gene 23 (gp23), the uncleaved form of the main shell protein, has been studied. Assembly and disassembly follow the predictions for entropy-driven processes; assembly is strongly favored by conditions of high salt concentrations and high temperatures, whereas low salt and low temperatures promote disassembly. In the absence of the scaffolding core proteins in vitro, only polyheads, the tubular variant of the prohead, are produced. Kinetic studies show that the rate of polyhead dissociation depends on the concentration of associated protein, not on the number and length of the particles. Comparable to crystal formation, assembly of gp23 occurs above a critical concentration, which is dependent on salt concentration, pH and temperature. These characteristics are common to most self-assembling systems. The oligomeric states of gp23 have been investigated by analytical ultracentrifugation, which indicated the existence, at very low salt concentration and low temperature, of an equilibrium between monomers and higher oligomers, culminating in the hexamer. At pH 9.0 polyheads are completely dissociated into their monomeric gp23 subunits. Our data suggest that the hexamer is a true intermediate of polyhead assembly.     

Colony formation by subpopulations of human T lymphocytes. III. Antigenic phenotype and function of colony-forming cells, colony cells, and their expanded progeny

The antigenic phenotype of individual PHA-induced T lymphocyte colonies was studied with a direct immunofluorescence technique using fluorescein-labeled anti-Leu-2a and anti-Leu-3a antibodies. Of the colonies grown from mononuclear peripheral blood cells 85% were Leu-3a+ (inducer/helper phenotype), 12% were Leu-2a+ (suppressor/cytotoxic phenotype), and 3% contained equal numbers of Leu-2a+ and Leu-3a+ cells. Fluorescence-activated cell sorter (FACS) separated T-cell subsets showed that Leu-2a+ cells and Leu-3a+ cells form exclusively Leu-2a+ and Leu-3a+ colonies, respectively. Leu-3a+ cells formed colonies in both the absence and presence of conditioned medium (PHA-CM), whereas colony formation by Leu-2a+ cells was absolutely dependent on PHA-CM. Mixing experiments with FACS-separated T-cell subsets showed that Leu-2a+ cells inhibit colony formation by Leu-3a+ cells in a cell dose-dependent manner both in the presence and absence of PHA-CM. Phenotype analysis of individual colonies from mixing experiments strongly suggested monoclonal proliferation in the present colony assay system. The majority of expanded T-cell colonies showed helper activity in a reverse hemolytic plaque-forming B-cell assay, although to a lesser degree as compared to that of freshly isolated T lymphocytes.     

Selective regulation of acid-sensing ion channel 1 by serine proteases

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that belong to the epithelial Na(+) channel/degenerin family. ASICs are transiently activated by a rapid drop in extracellular pH. Conditions of low extracellular pH, such as ischemia and inflammation in which ASICs are thought to be active, are accompanied by increased protease activity. We show here that serine proteases modulate the function of ASIC1a and ASIC1b but not of ASIC2a and ASIC3. We show that protease exposure shifts the pH dependence of ASIC1a activation and steady-state inactivation to more acidic pH. As a consequence, protease exposure leads to a decrease in current response if ASIC1a is activated by a pH drop from pH 7.4. If, however, acidification occurs from a basal pH of approximately 7, protease-exposed ASIC1a shows higher activity than untreated ASIC1a. We provide evidence that this bi-directional regulation of ASIC1a function also occurs in neurons. Thus, we have identified a mechanism that modulates ASIC function and may allow ASIC1a to adapt its gating to situations of persistent extracellular acidification.