The genetic characteristics Saccharomyces cerevisiae aci(+) mutants

A series of 30 Saccharomyces cerevisiae aci(+) mutants (characterized as acidifying Ogur's glucose medium containing bromocresol purple) were isolated after EMS mutagenesis. All the mutants excreted acid metabolites to the medium after 24 or 48 hours of incubation. The character of the aci(+) mutations was defined using classical genetic techniques. Three of the aci(+) mutants were studied by molecular genetics techniques.     

The MADS-Domain Factors AGAMOUS-LIKE15 and AGAMOUS-LIKE18, along with SHORT VEGETATIVE PHASE and AGAMOUS-LIKE24, Are Necessary to Block Floral Gene Expression during the Vegetative Phase

Multiple factors, including the MADS-domain proteins AGAMOUS-LIKE15 (AGL15) and AGL18, contribute to the regulation of the transition from vegetative to reproductive growth. AGL15 and AGL18 were previously shown to act redundantly as floral repressors and upstream of FLOWERING LOCUS T (FT) in Arabidopsis (Arabidopsis thaliana). A series of genetic and molecular experiments, primarily focused on AGL15, was performed to more clearly define their role. agl15 agl18 mutations fail to suppress ft mutations but show additive interactions with short vegetative phase (svp) mutations in ft and suppressor of constans1 (soc1) backgrounds. Chromatin immunoprecipitation analyses with AGL15-specific antibodies indicate that AGL15 binds directly to the FT locus at sites that partially overlap those bound by SVP and FLOWERING LOCUS C. In addition, expression of AGL15 in the phloem effectively restores wild-type flowering times in agl15 agl18 mutants. When agl15 agl18 mutations are combined with agl24 svp mutations, the plants show upward curling of rosette and cauline leaves, in addition to early flowering. The change in leaf morphology is associated with elevated levels of FT and ectopic expression of SEPALLATA3 (SEP3), leading to ectopic expression of floral genes. Leaf curling is suppressed by sep3 and ft mutations and enhanced by soc1 mutations. Thus, AGL15 and AGL18, along with SVP and AGL24, are necessary to block initiation of floral programs in vegetative organs.Appropriate timing of the shift from vegetative to reproductive growth is an important determinant of plant fitness. The time at which a plant flowers is determined through integration of signals reflecting extrinsic and intrinsic conditions, such as photoperiod, the duration of cold, plant health, and age (for review, see Amasino, 2010). One of the most important pathways regulating the timing of the floral transition is the photoperiod pathway (for review, see Imaizumi and Kay, 2006). Under long-day (LD) inductive conditions in Arabidopsis (Arabidopsis thaliana), photoperiod pathway components act to promote flowering by inducing CONSTANS (CO) and downstream genes. The floral integrator FLOWERING LOCUS T (FT) is a major target of multiple flowering pathways and the photoperiod pathway in particular. It is directly activated by CO (Samach et al., 2000). Under LD conditions, the peak of CO expression is coincident with the presence of light, and CO activates FT expression in the leaf vascular system (Yanovsky and Kay, 2003). FT travels through the phloem to the shoot apex (Corbesier et al., 2007), where, together with FLOWERING LOCUS D (Abe et al., 2005; Wigge et al., 2005), it activates APETALA1 (AP1) and other floral meristem identity genes, starting the flowering process. Other flowering time pathways converge on FT and/or directly impact gene expression in the meristem. The changes in gene expression that accompany the floral transition must be rapid, robust, largely irreversible, and strictly controlled spatially. This is achieved through positive feed-forward and negative feedback loops involving multiple regulatory factors (for recent review, see Kaufmann et al., 2010).Members of the MADS-box family of regulatory factors are central players in the regulatory loops controlling the floral transition (for a recent review, see Smaczniak et al., 2012a). MADS-domain factors typically act in large multimeric complexes and are well suited for regulation that involves combinatorial action. During the floral transition, MADS-domain proteins can act either as repressors or activators. In Arabidopsis, important floral repressors include SHORT VEGETATIVE PHASE (SVP) and members of the FLOWERING LOCUS C (FLC)-like group, including FLC, FLOWERING LOCUS M (FLM)/MADS AFFECTING FLOWERING1 (MAF1), and MAF2 to MAF5. Promoters of flowering include such MADS-domain factors as SUPPRESSOR OF CONSTANS1 (SOC1) and AGAMOUS-LIKE24 (AGL24). Together with non-MADS-box proteins FT and TWIN SISTER OF FT, SOC1 and AGL24 function as floral integrators. These operate downstream of the flowering time pathways but upstream of the meristem identity regulators such as LEAFY (LFY) and the MADS-domain factor AP1.The MADS-domain factors AGL15 and AGL18 also contribute to regulation of the floral transition in Arabidopsis. While single mutants have no phenotype, agl15 agl18 double mutants flower earlier than the wild type (Adamczyk et al., 2007). Therefore, AGL15 and AGL18 appear to act in a redundant fashion in seedlings, and like SVP, FLC, and MAF1 to MAF5, they act as floral repressors. The contributions of AGL15 and AGL18 are most apparent in the absence of strong photoperiodic induction: the agl15 agl18 double mutant combination partially suppresses the delay in flowering observed in co mutants, as well as the flowering delay associated with growth under short-day (SD) noninductive conditions. The earlier flowering in agl15 agl18 mutants under these conditions is associated with up-regulation of FT, and both AGL15 and AGL18 are expressed in the vascular system and shoot apex of young seedlings (Adamczyk et al., 2007), raising the possibility that AGL15 and AGL18 act directly on FT in leaves, as well as other targets in the meristem.AGL15, and to a lesser extent AGL18, have been further implicated in the networks that control flowering through molecular studies. Zheng et al. (2009) performed a chromatin immunoprecipitation (ChIP) analysis using AGL15-specific antibodies, tissue derived from embryo cultures, and a tiling array. Floral repressors (SVP and FLC), floral integrators (FT and SOC1), and a microRNA targeting AP2-like factors (miR172a) were identified as possible AGL15 targets (Zheng et al., 2009), suggesting that AGL15 may contribute to regulation through multiple avenues during the floral transition. AGL15 itself is directly bound and activated by AP2, which is both an A-class floral identity gene and a floral repressor (Yant et al., 2010). AGL15 is down-regulated in ap2 mutants, which are early flowering, while AGL18 is the nearest locus to multiple AP2-bound sites (Yant et al., 2010). Both AGL15 and AGL18 were identified as SOC1 targets through ChIP analyses (Immink et al., 2009; Tao et al., 2012). In yeast (Saccharomyces cerevisiae) two-hybrid assays, AGL15 interacts with a number of other MADS-domain proteins (de Folter et al., 2005), and in a one-hybrid study based on the SOC1 promoter, AGL15-SVP, AGL15-AGL24, and AGL15-SOC1 heterodimers were shown to bind to regions containing CArG boxes (Immink et al., 2012). AGL18 may act redundantly to AGL15 in these contexts. However, AGL18 either does not interact or only interacts weakly with other proteins in yeast two-hybrid assays (de Folter et al., 2005; Hill et al., 2008; Causier et al., 2012). It remains to be determined whether this truly reflects weaker or nonredundant in planta interactions or a technical problem in the artificial yeast system.Guided by the knowledge gained about AGL15 targets and interactions from molecular studies, we asked the following question: what is the functional significance of these molecular relationships in the context of the floral transition? We performed a series of genetic experiments combining agl15 agl18 mutations and mutations in interacting factors such as SVP, AGL24, and SOC1, as well as targets such as FT and SOC1. We also performed further molecular experiments focused on AGL15, for which a variety of tools are available. Among other things, we show that AGL15 and AGL18, along with AGL24 and SVP, play a role in blocking expression of the floral MADS-domain factor SEPALLATA3 (SEP3) during the vegetative phase. In the absence of these four factors, reproductive programs are initiated early, and floral genes are expressed in the youngest rosette leaf and cauline leaves.     

Haldane rules: costs of outbreeding at production of daughters in sand lizards

Haldane's rule is one of the most widely applicable paradigms in evolutionary biology, stating that in species crossings, the heterogametic sex will suffer more severely in terms of sterility and inviability. We address this in a within‐species outbreeding situation by assessing the risk of producing inviable offspring depending on the sex ratio of the clutch produced in between‐population crossings in the laboratory. In crossings between male and female sand lizards (Lacerta agilis) from two different sampling regions, one in Sweden, one in central Europe, risk of gametic incompatibility is unaffected by outbreeding, but offspring from between‐population crossings show 300% higher malformation frequency and 10% lower hatching success. The risk of having inviable offspring increases with the production of daughters, i.e. the hemizygous sex in this species (ZW). Such sex‐specific genetic costs of offspring production need to be incorporated into life history ecology, e.g. sex allocation theory.     

Accumulation of red elemental selenium nanoparticles and their biological effects in Nicotinia tabacum

The uptake, accumulation and biological effects of red nano-sized elemental selenium (nanoSe) in comparison to selenate were investigated in plant system at the first time. The data clearly indicated that red nanoSe was taken up by tobacco callus cultures and rooted tobacco plantlets. The roots of regenerated plantlets accumulated selenium in very high concentrations, 2,947 ± 99 mg/kg DW, from the medium containing 530 μM nanoSe. The biological effects of nanoSe were different from the selenate ion in plant tissue culture. NanoSe (265–530 μM concentration range) stimulated the organogenesis and the growth of root system significantly (~40 %) while selenate did not show these effects at any concentration moreover inhibited both callus growth and root regeneration totally in 265–530 μM concentrations.     

Proteins reacting with anti-spectrin antibodies are present in Chlamydomonas Cells.

It was found either in Western-blot analysis or in indirect immunofluorescence microscopy that cells of the alga Chlamydomonas reinhardhi contain polypeptides cross-reactng with antibodies directed against red blood cell spectrin. The protein could also be detected by immunoprecipitation with anti-spectrin antibodies. C. reinhardtii cells contain distinct polypeptide chains reacting with antibodies directed against either α- or β- spectrin subunits. This protein was extracted from the cells with low ionic strength solution but was not with nonionic detergent.     

Avidin, a potential biopesticide and synergist to Bacillus thuringiensis toxins against field crop insects

A meridic diet was supplemented with avidin at various concentrations to determine its effects on growth and mortality of three lepidopteran insects: Helicoverpa zea (Boddie), Spodoptera exigua (Hübner), and Anticarsia gemmatalis (Hübner). All insects were placed on diet immediately after hatching and observed until death or pupation occurred. At a concentration of 10 ppm, avidin had little or no effect on growth and mortality compared with the control. However at a concentration of 100 ppm almost all tested insects were killed. H. zea was further tested by adding sublethal concentrations of Bt (CrylAc) in the diet containing avidin. The synergistic effect was significant, with mortality increasing to 44.4% over additive mortality (21.6%) of Bt and avidin.     

The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions

In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate–bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA–MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein–cytoskeleton interactions are a universally conserved feature.     

Modulation of Glutamate Transport and Receptor Binding by Glutamate Receptor Antagonists in EAE Rat Brain

The etiology of multiple sclerosis (MS) is currently unknown. However, one potential mechanism involved in the disease may be excitotoxicity. The elevation of glutamate in cerebrospinal fluid, as well as changes in the expression of glutamate receptors (iGluRs and mGluRs) and excitatory amino acid transporters (EAATs), have been observed in the brains of MS patients and animals subjected to experimental autoimmune encephalomyelitis (EAE), which is the predominant animal model used to investigate the pathophysiology of MS. In the present paper, the effects of glutamatergic receptor antagonists, including amantadine, memantine, LY 367583, and MPEP, on glutamate transport, the expression of mRNA of glutamate transporters (EAATs), the kinetic parameters of ligand binding to N-methyl-D-aspartate (NMDA) receptors, and the morphology of nerve endings in EAE rat brains were investigated. The extracellular level of glutamate in the brain is primarily regulated by astrocytic glutamate transporter 1 (GLT-1) and glutamate-aspartate transporter (GLAST). Excess glutamate is taken up from the synaptic space and metabolized by astrocytes. Thus, the extracellular level of glutamate decreases, which protects neurons from excitotoxicity. Our investigations showed changes in the expression of EAAT mRNA, glutamate transport (uptake and release) by synaptosomal and glial plasmalemmal vesicle fractions, and ligand binding to NMDA receptors; these effects were partially reversed after the treatment of EAE rats with the NMDA antagonists amantadine and memantine. The antagonists of group I metabotropic glutamate receptors (mGluRs), including LY 367385 and MPEP, did not exert any effect on the examined parameters. These results suggest that disturbances in these mechanisms may play a role in the processes associated with glutamate excitotoxicity and the progressive brain damage in EAE.     

Application of surface plasmon resonance toward studies of low-molecular-weight antigen-antibody binding interactions

Methods for studying low-molecular-weight antigen-antibody binding interactions using surface plasmon resonance detection are presented. The experimental parameters most relevant to studies of low-molecular-weight antigen-antibody binding interactions are discussed. Direct kinetic analysis of the binding interactions is most informative, providing both apparent association and dissociation rate constants from which equilibrium constants can be calculated. Equilibrium analysis, including steady-state and solution affinity studies, offers an alternative approach to direct kinetic analysis when knowledge of the individual kinetic rate constants is not required or difficult to determine. The various methods are illustrated by studies of an anti-T(4) Fab fragment binding interaction with several thyroxine analogs. The methods utilized were dependent on the affinity of the interaction. The high-affinity anti-T(4) Fab fragment/l-T(4) binding interaction was evaluated using direct kinetic analysis. An intermediate affinity anti-T(4) Fab fragment/l-T(3) binding interaction was evaluated using a combination of direct kinetic analysis, steady-state analysis, and solution affinity analysis. The relatively weak anti-T(4) Fab fragment/l-T(2) binding interaction was evaluated using steady-state and solution affinity analysis protocols. Several thyroxine tracers that could not be immobilized to a biosensor surface were also evaluated via the solution affinity format. In cases where a given binding interaction was examined using multiple methods the results were comparable.     

CNPase activity in myelin from adult rat brains after prolonged lead exposure in vivo

The present study investigated the sensitivity of rats cerebral myelin to prolonged toxicity of lead (Pb) that imitates environmental exposure to this metal. The results indicated that 90 days exposure of young adult rats to lead in drinking water affects the morphology of myelin sheaths, expressed in disintegration of its multilamellar structure. Both, the protein content and the activity of the myelin-specific enzyme CNPase (2',3'-cyclic nucleotide 3-phosphodiesterase), were lowered. The Michaelis-Menten kinetic for CNPase in myelin obtained from control and Pb-treated rats was different. Km increased and Vmax decreased when compared to controls. Observed disturbances in enzyme activity may be one of the potential reasons of the ultrastructural changes. It is thus tempting to speculate that Pb may be considered as a one of the factors contributing to demyelinating diseases.