Stomatal responses of Vicia faba L. to indole acetic acid and abscisic acid

Evidence is presented that stomata in isolated epidermal peelsof Vicia faba L. open in darkness in response to the externalpresence of indole acetic acid (IAA) in the incubation medium.The effect of IAA is found to be overcome completely in thepresence of either TRIS or MES buffers. In the absence of buffer,V. faba stomata are shown to be influenced by IAA in a concentration-dependenttrend which reached a maximum at an [IAA] of 10^–3 molm^–3. Further investigations reveal that stomata in thisspecies can be shown to respond to the presence of IAA and anotherphytohormone, abscisic acid (ABA). IAA and ABA are demonstratedto be antagonistic in their effects provided the incubationconditions are suitable. The data are discussed in relationto stomatal responses of other species in different treatmentconditions. Recommendations are made with respect to standardizationof incubation media during epidermal peel experiments. Key words: Vicia faba, stomata, indole acetic acid, abscisic acid, buffers     

CENP-C functions in centromere assembly,the maintenance of CENP-A asymmetry and epigenetic age in Drosophila germline stem cells

Germline stem cells divide asymmetrically to produce one new daughter stem cell and one daughter cell that will subsequently undergo meiosis and differentiate to generate the mature gamete. The silent sister hypothesis proposes that in asymmetric divisions, the selective inheritance of sister chromatids carrying specific epigenetic marks between stem and daughter cells impacts cell fate. To facilitate this selective inheritance, the hypothesis specifically proposes that the centromeric region of each sister chromatid is distinct. In Drosophila germ line stem cells (GSCs), it has recently been shown that the centromeric histone CENP-A (called CID in flies)—the epigenetic determinant of centromere identity—is asymmetrically distributed between sister chromatids. In these cells, CID deposition occurs in G₂ phase such that sister chromatids destined to end up in the stem cell harbour more CENP-A, assemble more kinetochore proteins and capture more spindle microtubules. These results suggest a potential mechanism of ‘mitotic drive’ that might bias chromosome segregation. Here we report that the inner kinetochore protein CENP-C, is required for the assembly of CID in G₂ phase in GSCs. Moreover, CENP-C is required to maintain a normal asymmetric distribution of CID between stem and daughter cells. In addition, we find that CID is lost from centromeres in aged GSCs and that a reduction in CENP-C accelerates this loss. Finally, we show that CENP-C depletion in GSCs disrupts the balance of stem and daughter cells in the ovary, shifting GSCs toward a self-renewal tendency. Ultimately, we provide evidence that centromere assembly and maintenance via CENP-C is required to sustain asymmetric divisions in female Drosophila GSCs.     

Calcium activation of heart mitochondrial oxidative phosphorylation: rapid kinetics of mVO2, NADH, AND light scattering

Parallel activation of heart mitochondria NADH and ATP production by Ca(2+) has been shown to involve the Ca(2+)-sensitive dehydrogenases and the F(0)F(1)-ATPase. In the current study we hypothesize that the response time of Ca(2+)-activated ATP production is rapid enough to support step changes in myocardial workload ( approximately 100 ms). To test this hypothesis, the rapid kinetics of Ca(2+) activation of mV(O(2)), [NADH], and light scattering were evaluated in isolated porcine heart mitochondria at 37 degrees C using a variety of optical techniques. The addition of Ca(2+) was associated with an initial response time (IRT) of mV(O(2)) that was dose-dependent with a minimum IRT of 0.27 +/- 0.02 s (n = 41) at 535 nm Ca(2+). The IRTs for NADH fluorescence and light scattering in response to Ca(2+) additions were similar to mV(O(2)). The Ca(2+) IRT for mV(O(2)) was significantly shorter than 1.6 mm ADP (2.36 +/- 0.47 s; p < or = 0.001, n = 13), 2.2 mm P(i) (2.32 +/- 0.29, p < or = 0.001, n = 13), or 10 mm creatine (15.6.+/-1.18 s, p < or = 0.001, n = 18) under similar experimental conditions. Calcium effects were inhibited with 8 microm ruthenium red (2.4 +/- 0.31 s; p < or = 0.001, n = 16) and reversed with EGTA (1.6 +/- 0.44; p < or = 0.01, n = 6). Estimates of Ca(2+) uptake into mitochondria using optical Ca(2+) indicators trapped in the matrix revealed a sufficiently rapid uptake to cause the metabolic effects observed. These data are consistent with the notion that extramitochondrial Ca(2+) can modify ATP production, via an increase in matrix Ca(2+) content, rapidly enough to support cardiac work transitions in vivo.     

Randomly organized lipids and marginally stable proteins: A coupling of weak interactions to optimize membrane signaling

Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cell's ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6–5.8 bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

A NASP (N1/N2)-related protein, Sim3, binds CENP-A and is required for its deposition at fission yeast centromeres

A defining feature of centromeres is the presence of the histone H3 variant CENP-A(Cnp1). It is not known how CENP-A(Cnp1) is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASP(Human) and N1/N2(Xenopus) and aligns with Hif1(S. cerevisiae), defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, yet it associates with CENP-A(Cnp1) and also binds H3. Cells defective in Sim3 function have reduced levels of CENP-A(Cnp1) at centromeres (and increased H3) and display chromosome segregation defects. Sim3 is required to allow newly synthesized CENP-A(Cnp1) to accumulate at centromeres in S and G2 phase-arrested cells in a replication-independent mechanism. We propose that one function of Sim3 is to act as an escort that hands off CENP-A(Cnp1) to chromatin assembly factors, allowing its incorporation into centromeric chromatin.     

Mutation of Semaphorin-6A disrupts limbic and cortical connectivity and models neurodevelopmental psychopathology

Psychiatric disorders such as schizophrenia and autism are characterised by cellular disorganisation and dysconnectivity across the brain and can be caused by mutations in genes that control neurodevelopmental processes. To examine how neurodevelopmental defects can affect brain function and behaviour, we have comprehensively investigated the consequences of mutation of one such gene, Semaphorin-6A, on cellular organisation, axonal projection patterns, behaviour and physiology in mice. These analyses reveal a spectrum of widespread but subtle anatomical defects in Sema6A mutants, notably in limbic and cortical cellular organisation, lamination and connectivity. These mutants display concomitant alterations in the electroencephalogram and hyper-exploratory behaviour, which are characteristic of models of psychosis and reversible by the antipsychotic clozapine. They also show altered social interaction and deficits in object recognition and working memory. Mice with mutations in Sema6A or the interacting genes may thus represent a highly informative model for how neurodevelopmental defects can lead to anatomical dysconnectivity, resulting, either directly or through reactive mechanisms, in dysfunction at the level of neuronal networks with associated behavioural phenotypes of relevance to psychiatric disorders. The biological data presented here also make these genes plausible candidates to explain human linkage findings for schizophrenia and autism.     

Vaccine-induced tumor-specific immunity despite severe B-cell depletion in mantle cell lymphoma

The role of B cells in T-cell priming is unclear, and the effects of B-cell depletion on immune responses to cancer vaccines are unknown. Although results from some mouse models suggest that B cells may inhibit induction of T cell-dependent immunity by competing with antigen-presenting cells for antigens, skewing T helper response toward a T helper 2 profile and/or inducing T-cell tolerance, results from others suggest that B cells are necessary for priming as well as generation of T-cell memory. We assessed immune responses to a well-characterized idiotype vaccine in individuals with severe B-cell depletion but normal T cells after CD20-specific antibody-based chemotherapy of mantle cell lymphoma in first remission. Humoral antigen- and tumor-specific responses were detectable but delayed, and they correlated with peripheral blood B-cell recovery. In contrast, vigorous CD4(+) and CD8(+) antitumor type I T-cell cytokine responses were induced in most individuals in the absence of circulating B cells. Analysis of relapsing tumors showed no mutations or change in expression of target antigen to explain escape from therapy. These results show that severe B-cell depletion does not impair T-cell priming in humans. Based on these results, it is justifiable to administer vaccines in the setting of B-cell depletion; however, vaccine boosts after B-cell recovery may be necessary for optimal humoral responses.     

A DNA polymerase alpha accessory protein, Mcl1, is required for propagation of centromere structures in fission yeast

Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1) kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+), which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.