Blood clam (Anadara inflata) red cells. partition in aqueoues two-polymer phase systems

Anadara inflata is a clam which has red blood cells in its hemolymph. Furthermore, the nucleated red blood cells contain two structurally distinct hemoglobins. Clam red cells were subjected to partition in aqueous dextran-polyethylene glycol two-phase systems with the following results:

1.

1. Clam red cells are the largest cells (about 20 μm in diameter) so far studied in two-polymer phases. It is shown that not only can such cells be partitioned in dextran-polyethylene glycol phase systems, but that countercurrent distribution resolves the clam red cell population into more and less metabolically active cells. The distribution of these cells in relation to the whole population is similar to that of young and old red cells from mammals.     

Dual Role of DNA in Regulating ATP Hydrolysis by the SopA Partition Protein

In bacteria, mitotic stability of plasmids and many chromosomes depends on replicon-specific systems, which comprise a centromere, a centromere-binding protein and an ATPase. Dynamic self-assembly of the ATPase appears to enable active partition of replicon copies into cell-halves, but for Walker-box partition ATPases the molecular mechanism is unknown. ATPase activity appears to be essential for this process. DNA and centromere-binding proteins are known to stimulate the ATPase activity but molecular details of the stimulation mechanism have not been reported. We have investigated the interactions which stimulate ATP hydrolysis by the SopA partition ATPase of plasmid F. By using SopA and SopB proteins deficient in DNA binding, we have found that the intrinsic ability of SopA to hydrolyze ATP requires direct DNA binding by SopA but not by SopB. Our results show that two independent interactions of SopA act in synergy to stimulate its ATPase. SopA must interact with (i) DNA, through its ATP-dependent nonspecific DNA binding domain and (ii) SopB, which we show here to provide an arginine-finger motif. In addition, the latter interaction stimulates ATPase maximally when SopB is part of the partition complex. Hence, our data demonstrate that DNA acts on SopA in two ways, directly as nonspecific DNA and through SopB as centromeric DNA, to fully activate SopA ATP hydrolysis.Faithful segregation of low copy number plasmids in bacteria depends on partition loci, named Par. Such loci are composed of two genes, generically termed parA and parB, encoding an ATPase and a DNA-binding protein, respectively, and a cis-acting centromeric site parS (reviewed in Ref. 1). These three essential elements are sufficient for the partition process. ParBs assemble on parS to form nucleoprotein structures called partition complexes (2–6). ParA ATPases are considered to be motors that direct displacement and positioning of partition complexes inside the cell.Partition systems have been classified into two major types, distinguished by the nature of their ATPase proteins (7). Type I is characterized by Walker box ATPases, which are specified by many plasmids and most bacterial chromosomes. In some (Type Ia) the nucleotide-binding P-loop is preceded by an N-terminal regulatory domain, in the others (Type Ib) it is not. Type II specifies actin-like ATPases and is present on relatively few plasmids. It is presently the best understood system at the molecular level (8–10). However, the underlying mechanism that drives partition still remains elusive for both systems. Our work aims at the understanding of an archetypal representative of Type Ia, namely SopABC of the Escherichia coli plasmid F.The several activities of Type Ia ParA proteins are regulated by binding of adenine nucleotides (11, 12), which induce conformational changes in the proteins (13, 14). In their apo and/or ADP-bound forms these proteins display site-specific DNA binding activity, recognizing their cognate promoters through their N-terminal domains. Such activity is involved in the autoregulation of par operon expression (15, 16). In the ATP-bound form, they specifically interact with cognate partition complexes through contact with ParB proteins. The ATP-bound form of type I ParAs spontaneously forms polymers, which appear as bundled filaments in electron micrographs (12, 17–19). The role of these filaments is not understood but they could be related to the rapid movement of partition complexes in the cell. In vivo, ParA proteins form dynamic assemblies that move back and forth in the cell if the cognate ParB protein and parS centromere are present (20–23). The link between this oscillatory behavior and the segregation of partition complexes is not clear. They both require the ATPase activity of ParA proteins but the role of ATP hydrolysis in the partition process is not understood.It has long been known that ParA partition proteins exhibit low intrinsic ATPase activity (24, 25). ATP hydrolysis is modestly stimulated by either DNA or the cognate ParB alone but is strongly activated (up to 35-fold) when both DNA and ParBs are present (12, 24, 25). The lack of major stimulation of ATPase by DNA in the absence of ParB proteins has been taken to mean that the DNA-bound form of ParB is the effective activator (26). However, incorporation of centromere sites in the DNA added to ParB did not increase stimulation of ATPase (24, 25), leaving doubts as to the role of the partition complex in ATPase activation.The mechanism by which ATP hydrolysis acts in the partition process is not known for type I systems. This is in marked contrast to actin-based partition ATPases whose ATPase activity is stimulated in growing filaments (8), where it provokes the rapid disassembly of filaments unless these are capped by the cognate partition complex (9). Therefore, for the type II partition system, ATP hydrolysis ensures discrimination between unproductive filaments that are rapidly disassembled and productive filaments that drive partition complexes to opposite ends of the cell. This dynamic instability, which ensures elongation of actin-like filaments only between two partition complexes to be segregated, thus provides regulation of the partition process.Recently, it has been shown that two members of the type I ParA family, Soj of Thermus thermophilus and SopA of plasmid F, bind nonspecific DNA in the presence of ATP (12, 26). Two studies revealed that this DNA binding activity is essential for partition (27, 28). Importantly, it has been shown that a SopA mutant deficient in DNA binding no longer stimulates ATP hydrolysis efficiently, suggesting that DNA could play a direct role in the regulation of the ATPase activity (28). This finding raises the issue of the interactions required for activation of the type I partition ATPase activity by cognate proteins and DNA.In this study, we have investigated the mechanism of activation of ATP hydrolysis by SopA. First, we have found that the formation of the F partition complex is required for strong stimulation of the SopA intrinsic ATPase activity. We have also found that the partition complex and DNA stimulate ATP hydrolysis independently but that these two independent interactions act in synergy to amplify SopA ATPase activity. Lastly, we have identified an arginine finger motif in SopB responsible for the stimulation of SopA ATPase activity.     

Insect herbivory on native and exotic aquatic plants: phosphorus and nitrogen drive insect growth and nutrient release

The invasive clam Corbicula fluminea causes severe environmental and economic impacts in invaded sites and fouled water-dependent industries. The biological control of invasive species has potential as an effective, safe and low-cost tool. The potential of using direct (clam consumption) and indirect approaches (predator-avoidance behaviour) to control the pest was evaluated, aiming at establishing a proof-of-principle regarding the suitability of such strategies in confined settings (e.g. pipelines, channels, raw water tanks and other water-dependent facilities). Invertebrate (Procambarus clarkii) and vertebrate (Lepomis gibbosus, Luciobarbus bocagei) predators were examined for their control potential. In feeding experiments, barbels foraged upon C. fluminea but ingested very few clams, and clam consumption by crayfish was only observed in the smallest size class (<1 cm). Additional experiments linked these results to the protective role of the clam’s shell. In practical terms, the potential of the fish species as C. fluminea predators is limited, but crayfish can be important predators of small clams. In a second set of experiments, putative predator avoidance by clams was assessed in dual-choice aquaria. Refuge availability, predator diet and light conditions were taken into account. Clam avoidance was observed under particular conditions, but the amplitude of dislocation was limited.     

The Effect of Five Biomass Cropping Systems on Soil-Saturated Hydraulic Conductivity Across a Topographic Gradient

Understanding the environmental impact of bioenergy crops is needed to inform bioenergy policy development. We determined the effects of five biomass cropping systems—continuous maize (Zea mays), soybean (Glycine max)-triticale (Triticosecale ×)/soybean-maize, maize-switchgrass (Panicum virgatum), triticale/sorghum (Sorghum bicolor), and triticale-aspen (Populus alba × P. grandidentata)—on soil-saturated hydraulic conductivity (K _S ) across a toposequence in central Iowa, USA. We compared data from the time of cropping system establishment in 2009 to 4 years post-establishment. Both our 2009 and 2013 data confirmed that cropping system impacts on K _S vary by landscape position. We found that differences in cropping system impacts were more likely to occur at lower landscape positions, specifically, within footslope and floodplain positions. Previous research on cropping system impacts suggested that grass and woody systems were associated with a general increase in K _S over time, with greater changes likely occurring at landscape positions with a higher erosive potential or lower SOC content. Our results confirmed that the triticale-aspen woody system was associated with a significant increase in K _S across all landscape positions. In contrast, we did not observe an increase in K _S under maize-switchgrass, which we attributed to the high density of switchgrass roots by the fourth year of study, but expect an increase in K _S under switchgrass under longer measurement periods. We also found a significant increase in K _S in the annual systems, likely due to the conversion to no-till soil management with cropping system establishment. We expect such differences to become more apparent over longer time scales as cropping systems continue to impact soil hydraulic properties.     

Molecular evidence of symbiotic activity between <Emphasis Type="Italic">Symbiodinium</Emphasis> and <Emphasis Type="Italic">Tridacna maxima</Emphasis> larvae

Dinoflagellates in the genus Symbiodinium (zooxanthellae) provide the photosynthesis that sustains the majority of primary production in coral reefs. They occur symbiotically with several phyla, including mollusks such as giant clams (Tridacna spp.). This mutualistic association is obligatory for the giant clams, but the exact point in which this symbiosis is established and the main translocated photosynthate are unknown. In this study, we tracked the expression of specific genes for symbiosis and glycerol synthesis during a time course experiment. Giant clam larvae were raised until 75 h post-fertilization and then infected with cultured isolates of Symbiodinium clade A3. Expression of symbiosis-specific and housekeeping genes was monitored at four time points. The expression of H⁺-ATPase, a symbiosis-specific gene in Symbiodinium, was observed at 24 h after symbiont acquisition by the clam larvae. The expression of an enzyme responsible for glycerol synthesis was also observed. Together, these results show that the symbiotic relationship was already in place 24 h after Symbiodinium acquisition, during veliger larval stage. This is the first report using a molecular symbiosis-specific marker that supports symbiotic activity between Symbiodinium and a metazoan larva of an organism that acquires symbionts horizontally. From the expression of the glycerol-synthesizing gene, it was qualitatively determined that Symbiodinium cells may produce glycerol regardless of whether they are free-living or in symbiosis.     

Immunolocalization of Non-Symbiotic Hemoglobins During Somatic Embryogenesis in Chicory

Hemoglobins are ancient O₂-binding proteins, ubiquitously found in eukaryotes. They have been categorized as symbiotic, nonsymbiotic and truncated hemoglobins. We have investigated the cellular localization of nonsymbiotic hemoglobin proteins during somatic embryogenesis in Cichorium hybrid leaves (Cichorium intybus L. var. sativum × C. endivia var. latifolia) using immunolocalization technique. These proteins were detected during the two steps of culture: induction and expression. In leaves, hemoglobins colocalised with plastids, which were dispersed in the parietal cytoplasm as well as in the two guard cells of a stomata, but not in epidermis cells. Upon induction of embryogenesis, in the dark, this pattern disappeared. During the induction phase, where competent cells reinitiate the cell cycle and prepare for mitosis, hemoglobins appeared initially near chloroplasts, and then in the vicinity of vascular vessels especially in the phloem and in cells surrounding the xylem vessels. When leaf fragments were transferred to another medium for the expression phase, hemoglobins were observed in the majority of the leaf blade cells and in small young embryos but not in the older ones. Hemoglobins were also detected in other leaves cells or tissues all along the process. The role of these nonsymbiotic hemoglobins during somatic embryogenesis is discussed.Key Words: chicory, immunolocalization, nonsymbiotic hemoglobin, somatic embryogenesis     

Heteromeric AtKC1��AKT1 Channels in Arabidopsis Roots Facilitate Growth under K+-limiting Conditions

Plant growth and development is driven by osmotic processes. Potassium represents the major osmotically active cation in plants cells. The uptake of this inorganic osmolyte from the soil in Arabidopsis involves a root K⁺ uptake module consisting of the two K⁺ channel α-subunits, AKT1 and AtKC1. AKT1-mediated potassium absorption from K⁺-depleted soil was shown to depend on the calcium-sensing proteins CBL1/9 and their interacting kinase CIPK23. Here we show that upon activation by the CBL·CIPK complex in low external potassium homomeric AKT1 channels open at voltages positive of E_K, a condition resulting in cellular K⁺ leakage. Although at submillimolar external potassium an intrinsic K⁺ sensor reduces AKT1 channel cord conductance, loss of cytosolic potassium is not completely abolished under these conditions. Depending on channel activity and the actual potassium gradients, this channel-mediated K⁺ loss results in impaired plant growth in the atkc1 mutant. Incorporation of the AtKC1 subunit into the channel complex, however, modulates the properties of the K⁺ uptake module to prevent K⁺ loss. Upon assembly of AKT1 and AtKC1, the activation threshold of the root inward rectifier voltage gate is shifted negative by approximately −70 mV. Additionally, the channel conductance gains a hypersensitive K⁺ dependence. Together, these two processes appear to represent a safety strategy preventing K⁺ loss through the uptake channels under physiological conditions. Similar growth retardation phenotypes of akt1 and atkc1 loss-of-function mutants in response to limiting K⁺ supply further support such functional interdependence of AKT1 and AtKC1. Taken together, these findings suggest an essential role of AtKC1-like subunits for root K⁺ uptake and K⁺ homeostasis when plants experience conditions of K⁺ limitation.Fundamental plant functions such as control of the membrane potential, osmo-regulation, and turgor-driven growth and movements are based on the availability to gain high cellular potassium concentrations (1). The absorption of this inorganic osmolyte from the soil by the root therefore represents a pivotal process for plant life. Classical experiments by Epstein et al. in 1963 (2) described K⁺ root uptake as a biphasic process mediated by two uptake mechanisms: high affinity potassium transport with apparent affinities of ∼20 μm and a low affinity transport system with K_m values in the millimolar range. During the last decades several molecular components of potassium transport systems have been identified and functionally characterized in plants (3, 4). Mutant analyses, heterologous expression, as well as radiotracer uptake experiments characterized the K⁺ channels AKT1·AtKC1 and members of the HAK·KT·KUP family as major components of the Arabidopsis thaliana root-localized potassium transport system (5–9). In this study we focused on AKT1 and AtKC1, members of the Arabidopsis Shaker-like K⁺ channel family. AKT1 is a voltage-dependent inward-rectifying K⁺ channel mediating potassium uptake over a wide range of external potassium concentrations (10–15). Root cells of the akt1-1 loss-of-function mutant completely lack inward rectifying K⁺ currents (12). As a consequence the growth of akt1-1 seedlings is strongly impaired on low potassium medium (100 μm and less) (11, 12, 15). Rescue of yeast growth on 20 μm K⁺ and patch clamp experiments (16, 17) directly demonstrated that plant inward rectifying K⁺ channels are capable of serving as high affinity potassium uptake transporters. AtKC1 shares its expression pattern with AKT1 (18–20). AtKC1 α-subunits, however, neither form functional channels in akt1-1 knock-out plants nor in heterologous expression systems. In contrast to root cells of akt1-1 loss of function mutants, root protoplasts of AtKC1 null mutants (atkc1-f) still exhibit inward rectifying potassium currents most likely derived from homomeric AKT1 tetramers (20). Inward K⁺ currents in this atkc1-f mutant were characterized by a more positive activation voltage. These data suggested that the AtKC1 α-subunits do not form K⁺ channels per se but modulate the properties of the AKT1·AtKC1 heterocomplex (20–22). Previously, two groups in their ground-breaking studies demonstrated that AKT1 is activated by the CBL²-interacting, serine/threonine kinase, CIPK23, particularly under low K⁺ conditions (23, 24). CIPK23 itself was shown to be activated by the two calcineurin B-like proteins, CBL1 and 9, acting in a Ca²⁺-dependent manner upstream of CIPK23 (25, 26). Genetic disruption of these elements resulted in transgenic plants exhibiting a phenotype comparable with that of the AKT1 loss of function mutant. This regulatory system, based on a calcium sensor, a protein kinase, and a K⁺ channel, was functionally reconstituted in Xenopus oocytes (23, 24, 27), suggesting that these elements are essential and sufficient to operate as a low K⁺-sensitive potassium uptake system. Here we report on the physiological properties of the heteromeric K⁺ uptake module formed by the predominant root potassium uptake channel subunits, AKT1 and AtKC1 and its regulating kinase complex, CBL1 and CIPK23. Our studies show that the physical interaction of the CBL1·CIPK23 complex is specific for AKT1 channels and does not involve the AtKC1 subunit. AKT1 possesses a K⁺ (absence) sensor affecting channel activity at submillimolar K⁺ concentrations by strongly reducing its maximal cord conductance. Despite this K⁺ sensor, upon activation, AKT1 homomeric channels were shown to represent a potassium leak at low external potassium concentrations. Integration of AtKC1 into the K⁺ uptake module, however, prevented potassium loss by modulating both the voltage sensor and conductance in the channel complex. Moreover, activation of the AKT1-like maize channel ZMK1 by CBL1·CIPK23 suggests a conserved interaction and regulation across monocot and dicotyledonous plant species. Our biophysical studies as well as growth assays with plant mutant lines lacking the respective channels underline that acquisition of potassium under limiting K⁺ conditions is mediated via the root AKT1·AtKC1 K⁺ uptake channel complex.     

<Emphasis Type="Italic">Volvox</Emphasis> (Chlorophyta,Volvocales) as a model organism in developmental biology

Model systems based on two or more related species with different types of development are finding increasing use in current comparative embryology. Green algae of the genus Volvox offer an interesting opportunity to study sex pheromones, morphogenesis as well as the formation of a somatic cell line undergoing terminal differentiation, senescence, and death as well as a line of reproductive cells, which at first grow and then undergo a series of consecutive divisions that give rise to new organisms. However, almost all studies of the recent years were conducted on a single species, Volvox carteri f. nagariensis. The goal of this publication was to advertise the cosmopolitan alga V. aureus as a model species in developmental biology. Published data on V. aureus are briefly reviewed in comparison with the development of V. carteri and outlooks of further studies are specified. In particular, the expediency of collecting new V. aureus strains from nature to study their development in clonal culture is outlined.     

<Emphasis Type="Italic">OsNHX2</Emphasis>, an Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene,can enhance salt tolerance in rice plants through more effective accumulation of toxic Na<Superscript>+</Superscript> in leaf mesophyll and bundle sheath cells

The Na⁺/H⁺ antiporters play an important role in salt tolerance in plants. However, the functions of OsNHXs in rice except OsNHX1 have not been well studied. Using the gain- and loss-of-function strategies, we studied the potential role of OsNHX2 in salt tolerance in rice. Overexpression of OsNHX2 (OsNHX2-OE) in rice showed the significant tolerance to salt stress than wild-type plants and OsNHX2 knockdown transgenic plants (OsNHX2-KD). Under salt treatments of 300-mM NaCl for 5 days, the plant fresh weights, relative water percentages, shoot heights, Na⁺ contents, K⁺ contents, and K⁺/Na⁺ ratios in leaves of OsNHX2-OE transgenic plants were higher than those in wild-type plants, while no differences were detected in roots. K⁺/Na⁺ ratios in rice leaf mesophyll cells and bundle sheath cells were higher in OsNHX2-OE transgenic plants than in wild-type plants and OsNHX2-KD transgenic plants. Our data indicate that OsNHX2 plays an important role in salt stress based on leaf mesophyll cells and bundle sheath cells and can be served in genetically engineering crop plants with enhanced salt tolerance.     

Ion-exchange properties of the cell walls isolated from suspension-cultured plant cells

Ion-exchange capacity of the cell walls isolated from suspension-cultured Panax japonicus, Polyscias filicifolia and Dioscorea deltoidea cells was analyzed at pH 2.8–12 and constant ionic strength (100 mM). The cell walls of all cultures contain three types of ion-exchange groups: primary amino groups (pK _a < 3), carboxyl groups of polygalacturonic acid (pK _a 3.71), and carboxyl groups of hydroxycinnamic acids (pK _a 7.62). Amount of primary amino groups ranges from 500 (D. deltoidea) to 710 (P. japonicus) µmol/g cell wall dry weight, carboxyl groups with pK _a 3.71—from 570 (D. deltoidea) to 670 (P. filicifolia), carboxyl groups with pK _a 7.62—from 270 (P. filicifolia) to 370 (P. japonicus) µmol/g cell wall dry weight. The comparison of the data obtained by elemental and functional analyses demonstrated that the cell walls of all cultures are characterized by high content of pectins (~40% by weight) and structural proteins (~17–30% by weight), but do not contain phenolic OH–groups, which presumably signifies the absence of lignin in them.     

Genome-Wide Analysis of Potassium Transport-Related Genes in Chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) and Their Role in Abiotic Stress Responses

Potassium is the most abundant inorganic cation that constitutes up to 10% of the total plant dry weight and plays a prominent role in plant growth and development. Plants exhibit a complex but highly organized system of channels and transporters, which are involved in absorption and distribution of K⁺ from soil to different parts of plants. In this study, we explored the K⁺ transport system in chickpea genome and identified 36 genes encoding potassium channels and transporters. The identified genes were further classified on the basis of their domain structure and conserved motifs. It includes K⁺ transporters (23 genes: 2 HKTs, 6 KEAs, and 15 KUP/HAK/KTs) and K⁺ channels (13 genes: 8 Shakers and 5 TPKs). Chromosomal localization of these genes demonstrated that various K⁺ transporters and channels are randomly distributed across all the eight chromosomes. Comparative phylogenetic analysis of K⁺ transport system genes from Arabidopsis thaliana, Glycine max, Medicago truncatula, and Oryza sativa revealed their strong conservation in different plant species. Similarly, gene structure analysis displayed conservation of family-specific intron/exon organization in the K⁺ transport system genes. Evolutionary analysis of these genes suggested the segmental duplication as principal route of expansion for this family in chickpea. Several abiotic stress-related cis-regulatory elements were also identified in promoter regions suggesting their role in abiotic stress tolerance. Expression analysis of selected genes under drought, heat, osmotic, and salt stress demonstrated their differential expression in response to these stresses. This signifies the importance of these genes in the modulation of stress response in chickpea. Present study provides the first insight into K⁺ transport system in chickpea and can serve as a basis for their functional analysis.     

Anecdotal,Historical and Critical Commentaries on Genetics: In the Beginning Was the Worm …

The replicative life span (RLS) of Saccharomyces cerevisiae has been established as a model for the genetic regulation of longevity despite the inherent difficulty of the RLS assay, which requires separation of mother and daughter cells by micromanipulation after every division. Here we present the mother enrichment program (MEP), an inducible genetic system in which mother cells maintain a normal RLS—a median of 36 generations in the diploid MEP strain—while the proliferative potential of daughter cells is eliminated. Thus, the viability of a population over time becomes a function of RLS, and it displays features of a survival curve such as changes in hazard rate with age. We show that viability of mother cells in liquid culture is regulated by SIR2 and FOB1, two opposing regulators of RLS in yeast. We demonstrate that viability curves of these short- and long-lived strains can be easily distinguished from wild type, using a colony formation assay. This provides a simplified screening method for identifying genetic or environmental factors that regulate RLS. Additionally, the MEP can provide a cohort of cells at any stage of their life span for the analysis of age-associated phenotypes. These capabilities effectively remove the hurdles presented by RLS analysis that have hindered S. cerevisiae aging studies since their inception 50 years ago.THE budding yeast Saccharomyces cerevisiae is a popular model system for studying fundamental processes of cellular aging (reviewed in Steinkraus et al. 2008). Analyses over the past 50 years have led to the idea that budding yeast can be used to study three types of cellular aging. Replicative aging describes the division potential of individual cells and relies on the asymmetric cell divisions of budding yeast that yield distinct mother and daughter cells. Replicative life span (RLS) is defined as the number of times an individual cell divides before it undergoes senescence (Mortimer and Johnston 1959). Chronological aging describes the capacity of cells in stationary phase (analogous to G₀ in higher eukaryotes) to maintain viability over time, which is assayed by their ability to reenter the cell cycle when nutrients are reintroduced (Longo et al. 1996). Finally, budding yeast have been used to study clonal senescence, which is analogous to the Hayflick limit imposed on mammalian tissue culture cells and characterized by a finite number of times a population of cells can divide. Although wild-type yeast populations do not senesce, this phenomenon has been observed in mutant strains such as those lacking telomerase components (Lundblad and Szostak 1989; Singer and Gottschling 1994).While genetic screens have been applied to examine clonal and chronological aging (Lundblad and Szostak 1989; Powers et al. 2006; Murakami et al. 2008), they have been limited in their application to studying replicative aging (Kaeberlein and Kennedy 2005; Kaeberlein et al. 2005b). This limitation arises from the arduous nature of isolating replicatively aged yeast cells. The current “gold standard” for isolating aged mother cells is by micromanipulation, where daughter cells are counted and removed after every division (Park et al. 2002). Although micromanipulation is currently the only method capable of accurately measuring RLS in yeast, it is severely constrained by the small number of cells that can be analyzed. Thus, genetic analysis of the regulation of RLS has been limited to a candidate gene approach (reviewed in Steinkraus et al. 2008).True genetic analysis of RLS will require large populations of aged cells. However, there are two confounding issues that make isolation of aged individuals difficult. First, single-cell pedigree analysis has shown that age-associated phenotypes, such as replicative life span potential, segregate asymmetrically between mother and daughter cells, rendering age-associated phenotypes nonheritable (Egilmez and Jazwinski 1989; Kennedy et al. 1994). Thus, daughter cells are generally “reset” to a young state with every generation. Second, when age is measured in terms of cell divisions, an unfractionated population is predominately young. The fraction of the population at an age of n cell divisions is ∼1/2ⁿ. Individual cells that reach the median RLS, which is ∼26 generations for haploid cells of the S288C strain background (Kaeberlein et al. 2005a), represent an insignificant fraction of the total population. In fact, it is unlikely that any cell reaches such an advanced age because nutrient depletion will limit the division potential of the population (Dickinson and Schweizer 1999).As an alternative to micromanipulation, methods were developed to isolate aged cells from liquid cultures (Smeal et al. 1996; Sinclair and Guarente 1997; Chen and Contreras 2007). However, due to the exponential growth of progeny cells, these populations are technically limited to 7–12 generations before nutrient depletion interferes with replicative aging. While sequential rounds of growth and purification are possible, the inability to continuously follow an undisturbed cohort of cells prevents the measurement of RLS by these methods. Instead, purification methods are primarily used for the examination of molecular changes associated with aging cells. Unfortunately, low yields and loss of viability due to purification methods diminish their utility for analyzing phenotypes that affect cells of advanced age. As an alternative to purification from natural populations, a strategy to genetically regulate the replicative capacity of daughter cells and avoid the limits imposed by exponential growth has been described (Jarolim et al. 2004). While this system effectively prevents division of daughter cells, it unintentionally decreases the median RLS of mother cells to four cell divisions, thus restricting its usefulness.Here we describe the development of a novel genetic selection against newborn daughter cells, the “mother enrichment program” (MEP), which restricts the replicative capacity of daughter cells while allowing mother cells to achieve a normal RLS. We demonstrate that upon induction of the selection, the viability of MEP strains growing in liquid culture is determined by the RLS of the initial population of mother cells. MEP cultures therefore allow the comparison of RLS between strains without the need for micromanipulation. Additionally, because MEP cultures are not subject to nutrient limitation, single-step affinity purification of aged cells can be achieved at any point during their life span. Together, these capabilities substantially resolve the technical hurdles that have made replicative aging studies in S. cerevisiae exceptionally challenging.     

Characterization of uptake and release processes ford- andl-aspartate in primary cultures of astrocytes and cerebellar granule cells

The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameK _m - andV _max -values in bouth astrocytes (l-aspartate:K _m 77 μM;V _max 11.8 nmol×min^?1×mg^?1;d-aspartate:K _m 83 μM;V _max 14.0 nmol×min^?1×mg^?1) and granule cells (l-aspartate:K _m 32 μM;V _max 2.8 nmol ×min^?1×mg^?1;d-aspartate:K _m 26 μM;V _max 3.0 nmol×min^?1×mg^?1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca²⁺-dependent, K⁺-induced release was found for both amino acids.     

Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) hydraulic conductivity links to leaf venation architecture under well-watered condition rather than PEG-induced water deficit

Higher plant hydraulic conductivity (K _plant) is vital for plant growth, especially under PEG-induced water deficit stress (PEG-IWDS). Leaf venation architecture is a key determinant of leaf hydraulic conductivity (K _leaf) and K _leaf is a major component of K _plant across different plant species. However, there is little information about (1) varietal difference in leaf vein development in cereal crops, such as rice plants; (2) the effects of PEG-IWDS on leaf vein development; (3) the coordination between leaf venation architecture and K _plant as well as K _leaf under PEG-IWDS. In the present study, widely cultivated eight rice cultivars were grown hydroponically under well-watered condition (WWC) and PEG-IWDS, simulated by adding 15 % (w/v) PEG6000. Leaf venation architecture, including total longitudinal leaf vein number, leaf vein numbers per unit width (LVNW), vein thickness and leaf mass per area, as well as K _plant and K _leaf were measured to address above-mentioned questions. The results showed that leaf venation architecture exhibited significant varietal differences and PEG-IWDS significantly increased LVNW while decreased vein thickness. PEG-IWDS suppressed both K _plant and K _leaf but the decrease was much higher in K _plant than K _leaf. There was a significant and positive correlation observed between LVNW and K _leaf under both WWC and PEG-IWDS but the correlation between LVNW and K _plant was only significant under WWC. K _leaf was significantly and positively correlated with K _plant under WWC but not under PEG-IWDS. It is concluded that K _leaf is a major determinant for K _plant under WWC but not under PEG-IWDS; therefore, breeding or selecting rice cultivars with high LVNW can improve shoot water supplement under WWC but not under PEG-IWDS condition.