Floral bud reversion in Impatiens balsamina under non-inductive photoperiods

Summary All floral buds of Impatiens balsamina plants exposed to 4 short-day (SD) cycles and then returned to long days reverted to vegetative growth. The same happened with the upper buds of plants receiving a larger number of SDs, even as many as 90 cycles. The reversal proceeded in a basipetal order. The number of floral buds and flowers increased, and their reversion to vegetative growth was delayed with increasing numbers of SD cycles. Depending upon the stage attained by the floral bud before the transfer of the plant to noninductive photoperiods one or more inner whorls of the flower were replaced by a vegetative apex. The tip of the placenta was able to resume vegetative growth even after the formation of fertile anthers and an ovary with abortive ovules, showing that the potentiality for reversion is maintained till quite late stages in floral bud development. Continuous exposure to SD cycles is required not only for the continued production of floral buds, but also for their development to mature flowers, indicating that the floral stimulus in this plant is not self-perpetuating.     

Investigation of the polymorphic ScaI site by a PCR-based assay at the human atrial natriuretic peptides (hANP) gene locus

The ScaI polymorphic site within the stop codon of the human atrial natriuretic peptides (hANP) gene was investigated in Mauritian Indian, black African and French Caucasian populations. A distinct distribution pattern is observed in these three populations.     

Functional and pathogenic differences of Th1 and Th17 cells in experimental autoimmune encephalomyelitis

Background

There is consensus that experimental autoimmune encephalomyelitis (EAE) can be mediated by myelin specific T cells of Th1 as well as of Th17 phenotype, but the contribution of either subset to the pathogenic process has remained controversial. In this report, we compare functional differences and pathogenic potential of “monoclonal” T cell lines that recognize myelin oligodendrocyte glycoprotein (MOG) with the same transgenic TCR but are distinguished by an IFN-γ producing Th1-like and IL-17 producing Th17-like cytokine signature.

Methods and Findings

CD4⁺ T cell lines were derived from the transgenic mouse strain 2D2, which expresses a TCR recognizing MOG peptide 35–55 in the context of I-A^b. Adoptive transfer of Th1 cells into lymphopenic (Rag2^−/−) recipients, predominantly induced “classic” paralytic EAE, whereas Th17 cells mediated “atypical” ataxic EAE in approximately 50% of the recipient animals. Combination of Th1 and Th17 cells potentiated the encephalitogenicity inducing classical EAE exclusively. Th1 and Th17 mediated EAE lesions differed in their composition but not in their localization within the CNS. While Th1 lesions contained IFN-γ, but no IL-17 producing T cells, the T cells in Th17 lesions showed plasticity, substantially converting to IFN-γ producing Th1-like cells. Th1 and Th17 cells differed drastically by their lytic potential. Th1 but not Th17 cells lysed autoantigen presenting astrocytes and fibroblasts in vitro in a contact-dependent manner. In contrast, Th17 cells acquired cytotoxic potential only after antigenic stimulation and conversion to IFN-γ producing Th1 phenotype.

Conclusions

Our data demonstrate that both Th1 and Th17 lineages possess the ability to induce CNS autoimmunity but can function with complementary as well as differential pathogenic mechanisms. We propose that Th17-like cells producing IL-17 are required for the generation of atypical EAE whereas IFN-γ producing Th1 cells induce classical EAE.     

An interleukin-6 ZnO/SiO(2)/Si surface acoustic wave biosensor

A novel high sensitivity ZnO/SiO(2)/Si Love mode surface acoustic wave (SAW) biosensor for the detection of interleukin-6 (IL-6), is reported. The biosensors operating at 747.7MHz and 1.586GHz were functionalized by immobilizing the monoclonal IL-6 antibody onto the ZnO biosensor surface both through direct surface adsorption and through covalent binding on gluteraldehyde. The morphology of the IL-6 antibody-protein complex was studied using scanning electron microscopy (SEM), and the mass of the IL-6 protein immobilized on the surface was measured from the frequency shift of the SAW resonator biosensor. The biosensor was shown to have extended linearity, which was observed to improve with higher sensor frequency and for IL-6 immobilization through the monoclonal antibody. Preliminary results of biosensor measurements of low levels of IL-6 in normal human serum are reported. The biosensor can be fully integrated with CMOS Si chips and developed as a portable real time detection system for the interleukin family of proteins in human serum.     

Kinetics of salt-dependent unfolding of [2Fe–2S] ferredoxin of <Emphasis Type="Italic">Halobacterium salinarum</Emphasis>

The [2Fe–2S] ferredoxin from the extreme haloarchaeon Halobacterium salinarum is stable in high (>1.5 M) salt concentration. At low salt concentration the protein exhibits partial unfolding. The kinetics of unfolding was studied in low salt and in presence of urea in order to investigate the role of salt ions on the stability of the protein. The urea dependent unfolding, monitored by fluorescence of the tryptophan residues and circular dichroism, suggests that the native protein is stable at neutral pH, is destabilized in both acidic and alkaline environment, and involves the formation of kinetic intermediate(s). In contrast, the unfolding kinetics in low salt exhibits enhanced rate of unfolding with increase in pH value and is a two state process without the formation of intermediate. The unfolding at neutral pH is salt concentration dependent and occurs in two stages. The first stage, involves an initial fast phase (indicative of the formation of a hydrophobic collapsed state) followed by a relatively slow phase, and is dependent on the type of cation and anion. The second stage is considerably slower, proceeds with an increase in fluorescence intensity and is largely independent of the nature of salt. Our results thus show that the native form of the haloarchaeal ferredoxin (in high salt concentration) unfolds in low salt concentration through an apparently hydrophobic collapsed form, which leads to a kinetic intermediate. This intermediate then unfolds further to the low salt form of the protein.     

Dual-Color Monitoring Overcomes the Limitations of Single Bioluminescent Reporters in Fast-Growing Microbes and Reveals Phase-Dependent Protein Productivity during the Metabolic Rhythms of Saccharomyces cerevisiae

Luciferase is a useful, noninvasive reporter of gene regulation that can be continuously monitored over long periods of time; however, its use is problematic in fast-growing microbes like bacteria and yeast because rapidly changing cell numbers and metabolic states also influence bioluminescence, thereby confounding the reporter''s signal. Here we show that these problems can be overcome in the budding yeast Saccharomyces cerevisiae by simultaneously monitoring bioluminescence from two different colors of beetle luciferase, where one color (green) reports activity of a gene of interest, while a second color (red) is stably expressed and used to continuously normalize green bioluminescence for fluctuations in signal intensity that are unrelated to gene regulation. We use this dual-luciferase strategy in conjunction with a light-inducible promoter system to test whether different phases of yeast respiratory oscillations are more suitable for heterologous protein production than others. By using pulses of light to activate production of a green luciferase while normalizing signal variation to a red luciferase, we show that the early reductive phase of the yeast metabolic cycle produces more luciferase than other phases.     

Characterization of the formation of amyloid protofibrils from barstar by mapping residue-specific fluorescence dynamics

The small protein barstar aggregates at low pH to form soluble oligomers, which can be transformed into fibrillar aggregates at an elevated temperature. To characterize structurally, with residue-specific resolution, the process of amyloid formation of barstar, as well as to monitor the increase in size that accompanies the aggregation process, time-resolved fluorescence anisotropy decay measurements have been introduced as a valuable probe. Seven different single-cysteine-containing mutant forms of barstar were made, to each of which a fluorophore was attached at the thiol group. The rotational dynamics of these seven fluorophores, as well as of the sole intrinsic tryptophan residue in the protein, were determined in the amyloid protofibrils formed, as well as in the soluble oligomers from which the protofibrils arise upon heating. Mapping of the fast rotational dynamics onto the sequence of the protein yields dynamic amplitude maps that allowed identification of the segments of the chain that possess local structure in the soluble oligomer and amyloid protofibrils. The patterns of these maps of the soluble oligomer and protofibrils are seen to be similar; and protofibrils display more local structure than do the soluble oligomers, at all residue positions studied. The observation that transformation from soluble oligomers to protofibrils does not perturb local structure significantly at eight different residue positions, suggests that the soluble oligomers transform directly into protofibrils, without undergoing drastic structural rearrangements.     

DNA haplotype-dependent differences in the amino acid sequence of debrisoquine 4-hydroxylase (CYP2D6): evidence for two major allozymes in extensive metabolisers

The molecular basis for DNA haplotype-dependent debrisoquine 4-hydroxylase (CYP2D6) expression was explored by sequencing all of the nine exons of the CYP2D6 gene. Two distinct exon sequence frameworks of the CYP2D6 gene were found, each associated with specific BamHI-defined DNA haplotypes of the CYP2D cluster. They corresponded to Arg²⁹⁶/Cys²⁹⁶ and Ser⁴⁸⁶/Thr⁴⁸⁶ amino acid polymorphisms in the CYP2D6 enzyme, and occurred in almost equal frequency among the Caucasians examined. These two major allozymes with amino acid differences in the presumed substrate recognition region and in the vicinity of the heme binding site could be the source of the observed DNA haplotype-dependent variation in phenotypic expression.     

Solvent-exposed tryptophans probe the dynamics at protein surfaces.

The dynamics of single tryptophan (W) side chain of protease subtilisin Carlsberg (SC) and myelin basic protein (MBP) were used for probing the surface of these proteins. The W side chains are exposed to the solvent, as shown by the extent of quenching of their fluorescence by KI. Time-resolved fluorescence anisotropy measurements showed that the rotational motion of W is completely unhindered in the case of SC and partially hindered in the case of MBP. The rotational correlation time (phi) associated with the fast local motion of W did not scale linearly with the bulk solvent viscosity (eta) in glycerol-water mixtures. In contrast, phi values of either W side chains in the denatured proteins or the free W scaled almost linearly with eta, as expected by the Stokes-Einstein relationship. These results were interpreted as indicating specific partitioning of water at the surface of the proteins in glycerol-water mixtures.