Notch signalling is needed to maintain, but not to initiate, the formation of prosensory patches in the chick inner ear

Notch signalling is well-known to mediate lateral inhibition in inner ear sensory patches, so as to generate a balanced mixture of sensory hair cells and supporting cells. Recently, however, we have found that ectopic Notch activity at an early stage can induce the formation of ectopic sensory patches. This suggests that Notch activity may have two different functions in normal ear development, acting first to promote the formation of the prosensory patches, and then later to regulate hair-cell production within the patches. The Notch ligand Serrate1 (Jag1 in mouse and humans) is expressed in the patches from an early stage and may provide Notch activation during the prosensory phase. Here, we test whether Notch signalling is actually required for prosensory patch development. When we block Notch activation in the chick embryo using the gamma-secretase inhibitor DAPT, we see a complete loss of prosensory epithelial cells in the anterior otocyst, where they are diverted into a neuroblast fate via failure of Delta1-dependent lateral inhibition. The cells of the posterior prosensory patch remain epithelial, but expression of Sox2 and Bmp4 is drastically reduced. Expression of Serrate1 here is initially almost normal, but subsequently regresses. The patches of sensory hair cells that eventually develop are few and small. We suggest that, in normal development, factors other than Notch activity initiate Serrate1 expression. Serrate1, by activating Notch, then drives the expression of Sox2 and Bmp4, as well as expression of the Serrate1 gene itself. The positive feedback maintains Notch activation and thereby preserves and perhaps extends the prosensory state, leading eventually to the development of normal sensory patches.     

Bub1 and Bub3 promote the conversion from monopolar to bipolar chromosome attachment independently of shugoshin

The eukaryotic spindle assembly checkpoint (SAC) delays anaphase in the presence of chromosome attachment errors. Bub3 has been reported to be required for SAC activity in all eukaryotes examined so far. We find that Bub3, unlike its binding partner Bub1, is not essential for the SAC in fission yeast. As Bub3 is needed for the efficient kinetochore localization of Bub1, and of Mad1, Mad2 and Mad3, this implies that most SAC proteins do not need to be enriched at the kinetochores for the SAC to function. We find that Bub3 is also dispensable for shugoshin localization to the centromeres, which is the second known function of Bub1. Instead, Bub3, together with Bub1, has a specific function in promoting the conversion from chromosome mono‐orientation to bi‐orientation.     

Meiosis: polo,FEAR and the art of dividing reductionally

Recent studies on the regulation of meiosis have uncovered new roles for old acquaintances: the polo-like kinase Cdc5 has been found to dictate proper kinetochore orientation during meiosis I, while the FEAR pathway is essential for some, but not all, aspects of meiosis I exit.     

Interactions between regulatory and catalytic subunits of the Candida albicans cAMP-dependent protein kinase are modulated by autophosphorylation of the regulatory subunit

The cAMP-dependent protein kinase (PKA) from Candida albicans is a tetramer composed of two catalytic subunits (C) and two type II regulatory subunits (R). To evaluate the role of a putative autophosphorylation site of the R subunit (Ser(180)) in the interaction with C, this site was mutated to an Ala residue. Recombinant wild-type and mutant forms of the R subunit were expressed in Escherichia coli and purified. The wild-type recombinant R subunit was fully phosphorylated by the purified C subunit, while the mutant form was not, confirming that Ser(180) is the target for the autophosphorylation reaction. Association and dissociation experiments conducted with both recombinant R subunits and purified C subunit showed that intramolecular phosphorylation of the R subunit led to a decreased affinity for C. This diminished affinity was reflected by an 8-fold increase in the concentration of R subunit needed to reach half-maximal inhibition of the kinase activity and in a 5-fold decrease in the cAMP concentration necessary to obtain half-maximal dissociation of the reconstituted holoenzyme. Dissociation of the mutant holoenzyme by cAMP was not affected by the presence of MgATP. Metabolic labeling of yeast cells with [(32)P]orthophosphate indicated that the R subunit exists as a serine phosphorylated protein. The possible involvement of R subunit autophosphorylation in modulating C. albicans PKA activity in vivo is discussed.     

Interactions of rotor subunits in the chloroplast ATP synthase modulated by nucleotides and by Mg2+

ATP synthases - rotary nano machines - consist of two major parts, F(O) and F(1), connected by two stalks: the central and the peripheral stalk. In spinach chloroplasts, the central stalk (subunits gamma, epsilon) forms with the cylinder of subunits III the rotor and transmits proton motive force from F(O) to F(1), inducing conformational changes of the catalytic centers in F(1). The epsilon subunit is an important regulator affecting adjacent subunits as well as the activity of the whole protein complex. Using a combination of chemical cross-linking and mass spectrometry, we monitored interactions of subunit epsilon in spinach chloroplast ATP synthase with III and gamma. Onto identification of interacting residues in subunits epsilon and III, one cross-link defined the distance between epsilon-Cys6 and III-Lys48 to be 9.4 A at minimum. epsilon-Cys6 was competitively cross-linked with subunit gamma. Altered cross-linking yields revealed the impact of nucleotides and Mg(2+) on cross-linking of subunit epsilon. The presence of nucleotides apparently induced a displacement of the N-terminus of subunit epsilon, which separated epsilon-Cys6 from both, III-Lys48 and subunit gamma, and thus decreasing the yield of the cross-linked subunits epsilon and gamma as well as epsilon and III. However, increasing concentrations of the cofactor Mg(2+) favoured cross-linking of epsilon-Cys6 with subunit gamma instead of III-Lys48 indicating an approximation of subunits gamma and epsilon and a separation from III-Lys48.     

Cavity spot of carrots: Interactions between the host and pathogen, related to the cell wall

This study investigates the structural aspects of cavity spot pathogenesis. Different Pythium spp. isolated from infected carrots, apples and melons were cultured on agar in Petri dishes and used for inoculation of uninfected carrots. Only slow-growing Pythium spp. (< 15 mm day^-1), such as P. violae and P. sulcatum caused cavity spot lesions. It is suggested that slow-growing species are able to penetrate, albeit slowly, into the plant tissue for 3 to 4 days before a hypersensitive reaction develops. Fast-growing species, however, did not cause lesions. Based on ultrastructural observations, we suggest that the following sequence of events occurs between the plant and the pathogen: The fungus infects the walls and grows for several days, during which time small amounts of wall-degrading enzymes are secreted. Phenylalanine ammonia lyase (PAL) activity and phenols increase linearly immediately upon inoculation. There was a lag phase of about 5 days before lignin began to increase linearly for about a month. Dissolution of wall components decreases the solute potential and water potential in the apoplast. Thus, water moves from the symplast into the apoplast, the turgor pressure gradually dissipates, and the cells shrink and eventually die.     

Transport of lipids by ABC proteins: Interactions and implications for cellular toxicity, viability and function

Members of the ATP-binding cassette (ABC) family of membrane-bound transporters are involved in multiple aspects of transport and redistribution of various lipids and their conjugates. Most ABC transporters localize to the plasma membrane; some are associated with liquid-ordered cholesterol-/sphingolipid-rich microdomains, and to a lesser extent the membranes of the Golgi and endoplasmic reticulum. Hence, ABC transporters are well placed to regulate plasma membrane lipid composition and the efflux and redistribution of structural phospholipids and sphingolipids during periods of cellular stress and recovery. ABC transporters can also modulate cellular sensitivity to extrinsic pro-apoptotic signals through regulation of sphingomyelin-ceramide biosynthesis and metabolism. The functionality of ABC transporters is, in turn, modulated by the lipid content of the microdomains in which they reside. Cholesterol, a major membrane microdomain component, is not only a substrate of several ABC transporters, but also regulates ABC activity through its effects on microdomain structure. Several important bioactive lipid mediators and toxic lipid metabolites are also effluxed by ABC transporters. In this review, the complex interactions between ABC transporters and their lipid/sterol substrates will be discussed and analyzed in the context of their relevance to cellular function, toxicity and apoptosis.     

Interactions between learning and evolution: the outstanding strategy generated by the Baldwin effect

The Baldwin effect is known as an possible interaction between learning and evolution, where individual lifetime learning can influence the course of evolution without using any Lamarckian mechanism. Our concern is to consider the Baldwin effect in dynamic environments, especially when there is no explicit optimal solution through generations and this solution depends only on interactions among agents. We adopted the iterated Prisoner's Dilemma as a dynamic environment, introduced phenotypic plasticity into its strategies, and conducted computational experiments, in which phenotypic plasticity is allowed to evolve. The Baldwin effect was observed in the experiments as follows: First, strategies with enough plasticity spread, which caused a shift from defect-oriented populations to cooperative populations. Second, these strategies were replaced by a strategy with a modest amount of plasticity generated by interactions between learning and evolution. By making three kinds of analysis, we have shown that this strategy provides outstanding performance in comparison with other deterministic strategies. Further experiments towards open-ended evolution have also been conducted so as to generalize our results.     

The Uptake of Nitrite by the Diatom Phaeodactylum: Interactions between Nitrite and Nitrate

Freshly harvested ammonium-grown cells of the diatom Phaeodactylumtricornutum cannot take up nitrite but they acquire the abilityto do so after a period (about 3 h) of nitrogen deprivationunder conditions that allow photosynthesis. Addition of cycloheximide(1.0 µg ml^–1) prevents development as does incubationin Na⁺ or K⁺-free medium. Nitrite uptake is dependent on thepresence of Na⁺ in the medium and is inhibited by ammonium andnitrite uptake but it is concluded that the inhibition of nitriteuptake by nitrate is not of the competitive type.     

Interactions among genomic structure, function, and evolution revealed by comprehensive analysis of the Arabidopsis thaliana genome

The genome in a higher organism consists of a number of types of nucleotide sequence-specialized components, with each having tens of thousands of members or elements. It is crucial for our understanding of how a genome as an entity is organized, functions, and evolves to determine how these components are organized in the genome and how they relate with each other; however, no such knowledge is available. Here, we report a comprehensive analysis of the organization and interaction of all 40 components constituting the genome of the plant model species, Arabidopsis thaliana, at the whole-genome and chromosome levels. The 40 components include (i) 6 genome structural components consisting of GC%, genes, retrotransposons, DNA transposons, simple repeats, and low complex repeats; (ii) 3 evolutionarily critical features consisting of recombination rate, nucleotide substitutions, and nucleotide insertions/deletions; and (iii) 31 categories of genes with different functions and numbers of functions. We show that the distributions of 39 of the 40 components of the genome (excepting GC%) deviate significantly from the random distribution model and different types of the genome components are significantly correlated. These results remained to be true even when the genomic regions, such as centromeric regions, where transposable and repeat elements are abundant were excluded from the analyses. These findings suggest that DNA molecules contained in the Arabidopsis genome are each organized and structured from their constituting components in an unambiguous manner and that different types of the components that constitute or characterize the genome interact. The analysis also showed that each chromosome consists of a similar set of the components at similar densities, suggesting that the unique organization and interaction pattern of the components in each chromosome may represent, at least in part, the identity of a chromosome or a genome at the genome level, thus partly accounting for the phenotypic variation among different species. The data also provide comprehensive and new insights into many phenomena significant in genome biology, with which we particularly discuss the variation of genetic recombination. The variation of genetic recombination rate along a chromosomal arm is shaped, not only by the distribution of simple repeats, retrotransposons, DNA transposons, and nucleotide substitutions, but also by the functions of genes contained, especially those with multiple functions, suggesting that variation of genetic recombination along a chromosomal arm is the result of interactions among the components constituting local genome structure, function, and evolution.     

Magnesium uptake by pancreatic islet cells is modulated by stimulators and inhibitors of the B-cell function

28Mg2+ uptake by rat islets was measured during incubation with various stimulators or inhibitors of insulin release. D-Glucose induced a dose-dependent increase in 28Mg2+ uptake after 10 min or 120 min. The threshold concentration was around 6 mM and the maximum effect was observed with 15-20 mM glucose. After 120 min 28Mg2+ uptake was also stimulated by the metabolized sugars mannose, N-acetylglucosamine or glyceraldehyde, was unaffected by the non-metabolized or poorly metabolized L-glucose, galactose, 3-O-methylglucose, 2-deoxyglucose, fructose or mannoheptulose and was inhibited by glucosamine. The effect of glucose was markedly impaired by mannoheptulose, glucosamine, aminooxyacetate and NH4Cl, but was only partially decreased by D600 or diazoxide, which were ineffective in a glucose-free medium. Tolbutamide or KCl slightly increased 28Mg2+ uptake. Alanine, leucine alone or with glutamine, and ketoisocaproate also stimulated 28Mg2+ uptake, whereas arginine and lysine decreased it. These changes in 28Mg2+ uptake, brought about by various modifiers of the B-cell function, are thus similar but not identical to the changes in Ca2+ uptake, and are not the consequence of insulin release. The stimulatory effect of glucose requires glucose metabolism by islet cells, but is only partially due to depolarization of the B-cell membrane.     

Multiple genetic pathways involving the Caenorhabditis elegans Bloom's syndrome genes him-6, rad-51, and top-3 are needed to maintain genome stability in the germ line

Bloom's syndrome (BS) is an autosomal-recessive human disorder caused by mutations in the BS RecQ helicase and is associated with loss of genomic integrity and an increased incidence of cancer. We analyzed the mitotic and the meiotic roles of Caenorhabditis elegans him-6, which we show to encode the ortholog of the human BS gene. Mutations in him-6 result in an enhanced irradiation sensitivity, a partially defective S-phase checkpoint, and in reduced levels of DNA-damage induced apoptosis. Furthermore, him-6 mutants exhibit a decreased frequency of meiotic recombination that is probably due to a defect in the progression of crossover recombination. In mitotically proliferating germ cells, our genetic interaction studies, as well as the assessment of the number of double-strand breaks via RAD-51 foci, reveal a complex regulatory network that is different from the situation in yeast. Although the number of double-strand breaks in him-6 and top-3 single mutants is elevated, the combined depletion of him-6 and top-3 leads to mitotic catastrophe concomitant with a massive increase in the level of double-strand breaks, a phenotype that is completely suppressed by rad-51. him-6 and top-3 are thus needed to maintain low levels of double-strand breaks in normally proliferating germ cells, and both act in partial redundant pathways downstream of rad-51 to prevent mitotic catastrophy. Finally, we show that topoisomerase IIIalpha acts independently during a late stage of meiotic recombination.     

Interactions between aluminium,phosphorus and pH in the response of barley to soil acidity

Summary Two acid soils showing different Al solubility as a function of pH were limed to a range of pH values (in 10^–2M CaCl₂) between 4.1 and 5.6. The apparent critical pH for the growth of barley in pots was 0.25 lower in the soil showing lower Al solubility. The addition of phosphate reduced exchangeable and soluble Al in the soils, and lowered the apparent critical pH by 0.35 while maintaining the difference between the soils. The Al concentration at the critical pH, measured after cropping to take account of the treatment effects on soil Al, also varied with soil and with phosphate addition. These apparent critical values of both pH and soluble Al varied linearly with available phosphate, over the range 18 to 73 mg P/kg soil, as follows: pH from 4.9 to 4.3; soluble Al, from 0.010 mM to 0.056 mM; and the soluble Ca/Al mole ratio, from 1270 to 214.     

Mitochondrial matters of the heart: a plethora of regulatory modes to maintain function for a long lifetime

The human/animal heart, comprised of cells called “myocytes” is an incredible organ that to remain beating must be fueled constantly via the hydrolysis of adenosine tri-phosphate (ATP). Deriving most of its ATP from mitochondrial oxidative phosphorylation (ox phos), and a smaller amount from “glycolysis”, i.e., glucose conversion to pyruvate or lactate, the heart helps in the delivery of oxygen (via hemoglobin) to every organ/tissue in our body. Then, the empty (deoxy) hemoglobin returns to load more oxygen and the journey to tissues is repeated 24 h a day, year after year, until “death do us part”. To support this essential “pumping” process the heart must work constantly, i.e., 70–80 years (life expectancy in the U.S.). This is a remarkable feat when compared with one of our most costly people-made technologies, i.e., automobiles (cars). In the past century, it was rare to see the family car survive more than 10–15 years unless it had been subjected to motor replacement surgery. Most were laid to rest at a much earlier age. Now, in this new millennium should a brilliant car manufacturer succeed in constructing a car engine as efficient as the human heart, each family member requiring a car would need only one per life time. With this in mind, one of the major future “matters of the heart” is to keep it pumping, not only for the current 70–80 year life span but much longer. To do this depends on, among other matters, the two processes noted above, i.e., oxidative phosphorylation and glycolysis. The former is strictly a mitochondrial process that works only in the presence of oxygen whereas glycolysis, dependent on mitochondrial bound hexokinase 2 (MB-HK-2), works either in the presence or absence of oxygen. In addition, the MB-HK 2 is anti-apoptotic and helps with other factors to retard cell death. Current estimates reveal that the human heart of an individual living 70–80 years will have undergone 2.5–3.0 billion beats, a feat that is energetically feasible only due to the heart cells’ (cardiomyocytes) large population of mitochondria with bound HK-2.     

Specific attachment of nuclear-mitotic apparatus protein to metaphase chromosomes and mitotic spindle poles: possible function in nuclear reassembly

NuMA protein is the largest, abundant, primate-specific chromosomal protein. The protein was purified from HeLa cells and monospecific monoclonal antibodies were prepared that react exclusively with NuMA protein in immunoblot analysis. These antibodies were used to define the intracellular location and properties of NuMA protein. Using indirect immunofluorescence, NuMA protein was detected only in the nucleus of interphase cells and on the chromosomes in mitotic cells. One class of monoclonal antibody called the 2E4-type antibody, caused NuMA protein (or a complex of proteins including NuMA) to be released from its binding site on metaphase or anaphase chromosomes. The separation of NuMA protein from chromosomes was observed either with the immunofluorescence assay or in electrophoretic analyses of proteins released from isolated metaphase chromosomes after reaction with 2E4 antibody. The immunofluorescence studies also showed that after release of the NuMA protein from chromosomes of metaphase or anaphase cells, the protein bound specifically to the polar region of the mitotic spindle. It was shown that exogenously added NuMA antigen/antibody complex bound only to the mitotic spindle poles of permeabilized primate cells and not to the spindle poles of other mammalian cells, thus demonstrating the specificity of the spindle-pole interaction. The antibody mediated transfer of NuMA from chromosomes to poles was blocked when the chromosomes were treated with cross-linking fixatives. Results suggest that the NuMA protein has specific attachment sites on both metaphase chromosomes and mitotic spindle poles (the site where post-mitotic nuclear assembly occurs). A model is proposed suggesting that a protein having such dual binding sites could function during nuclear reassembly to link mitotic chromosomes into the reforming nucleus.