Erythrocytes and age. Disintegration of erythrocytes by brilliant cresyl blue in correlation to age

The author describes changes in the disintegration of erythrocytes by brilliant cresyl blue in correlation to age, in rats aged 21, 42, 90-105, 340-360 and 690-720 days. The erythrocytes were incubated for 4 hours in an isotonic NaCl solution, in Krebs-Ringer solution and in each of these solutions plus brilliant cresyl blue. Disintegration in plain NaCl solution was found to be the greatest in the case of erythrocytes from 690- to 720-day-old rats. In the same solution plus brilliant cresyl blue, the rate of disintegration was very high in 21-day-old, 42-day-old and 690- to 720-day-old animals; at 90-105 days it was lower and at 340-360 days it was the lowest. Disintegration of erythrocytes in plain Krebs-Ringer solution was the lowest at 21 and 42 days; in the other age groups it was slightly higher. On adding brilliant cresyl blue, the rate of disintegration rose significantly in 21-, 42- and 690- to 720-day-old animals; at 90-105 days and 340-360 days it was no different from disintegration in plain Krebs-Ringer solution. It can be seen from the results that the rate of brilliant cresyl blue-induced erythrocyte disintegration is dependent on the age of the animals from which the erythrocytes are taken.     

Reconstructing Native American Migrations from Whole-Genome and Whole-Exome Data

There is great scientific and popular interest in understanding the genetic history of populations in the Americas. We wish to understand when different regions of the continent were inhabited, where settlers came from, and how current inhabitants relate genetically to earlier populations. Recent studies unraveled parts of the genetic history of the continent using genotyping arrays and uniparental markers. The 1000 Genomes Project provides a unique opportunity for improving our understanding of population genetic history by providing over a hundred sequenced low coverage genomes and exomes from Colombian (CLM), Mexican-American (MXL), and Puerto Rican (PUR) populations. Here, we explore the genomic contributions of African, European, and especially Native American ancestry to these populations. Estimated Native American ancestry is in MXL, in CLM, and in PUR. Native American ancestry in PUR is most closely related to populations surrounding the Orinoco River basin, confirming the Southern America ancestry of the Taíno people of the Caribbean. We present new methods to estimate the allele frequencies in the Native American fraction of the populations, and model their distribution using a demographic model for three ancestral Native American populations. These ancestral populations likely split in close succession: the most likely scenario, based on a peopling of the Americas thousand years ago (kya), supports that the MXL Ancestors split kya, with a subsequent split of the ancestors to CLM and PUR kya. The model also features effective populations of in Mexico, in Colombia, and in Puerto Rico. Modeling Identity-by-descent (IBD) and ancestry tract length, we show that post-contact populations also differ markedly in their effective sizes and migration patterns, with Puerto Rico showing the smallest effective size and the earlier migration from Europe. Finally, we compare IBD and ancestry assignments to find evidence for relatedness among European founders to the three populations.     

Carbon flux analysis in a pantothenate overproducing Corynebacterium glutamicum strain

Carbon flux analysis during a pseudo-stationary phase of metabolite accumulation in a genetically engineered strain of Corynebacterium glutamicum, containing plasmids leading to over-expression of the ilvBNCD and panBC operons, has identified the basic metabolic constraints governing the potential of this bacterium to produce pantothenate. Carbon flux converging on pyruvate (75% of glucose uptake) is controlled by anabolic precursor requirements and NADPH demand provoking high carbon loss as CO₂ via the pentose pathway. Virtually all the flux of pyruvate is directed into the branched pathway leading to both valine and pantothenate production, but flux towards valine is tenfold higher than that transformed to pantothenate, indicating that significant improvements will only be obtained if carbon flux at the ketoisovalerate branchpoint can be modulated.     

Development of a phenomenological modeling approach for prediction of growth and xanthan gum production using Xanthomonas campestris

An unstructured kinetic model for xanthan production is described and fitted to experimental data obtained in a stirred batch reactor. The culture medium was composed of several nitrogen sources (soybean hydrolysates, ammonium and nitrate salts) consumed sequentially. The model proposed is able to describe this sequential consumption of nitrogen sources, the consumption of inorganic phosphate and carbon, the evolution of biomass, and production of xanthan. The parameter estimation has been performed by fitting the kinetic model in differential form to experimental data. Runs of the model for simulating xanthan gum production as a function of the initial concentration of inorganic phosphate have shown the positive effect of phosphate limitation on xanthan yield, though diminishing rates of production. The model was used to predict the kinetic parameters for a medium containing a 2-fold lower initial phosphate concentration. When tested experimentally, the measured fermentation parameters were in close agreement with the predicted model values, demonstrating the validity of the model.     

The Role of Edaphic Environment and Climate in Structuring Phylogenetic Pattern in Seasonally Dry Tropical Plant Communities

Seasonally dry tropical plant formations (SDTF) are likely to exhibit phylogenetic clustering owing to niche conservatism driven by a strong environmental filter (water stress), but heterogeneous edaphic environments and life histories may result in heterogeneity in degree of phylogenetic clustering. We investigated phylogenetic patterns across ecological gradients related to water availability (edaphic environment and climate) in the Caatinga, a SDTF in Brazil. Caatinga is characterized by semiarid climate and three distinct edaphic environments – sedimentary, crystalline, and inselberg –representing a decreasing gradient in soil water availability. We used two measures of phylogenetic diversity: Net Relatedness Index based on the entire phylogeny among species present in a site, reflecting long-term diversification; and Nearest Taxon Index based on the tips of the phylogeny, reflecting more recent diversification. We also evaluated woody species in contrast to herbaceous species. The main climatic variable influencing phylogenetic pattern was precipitation in the driest quarter, particularly for herbaceous species, suggesting that environmental filtering related to minimal periods of precipitation is an important driver of Caatinga biodiversity, as one might expect for a SDTF. Woody species tended to show phylogenetic clustering whereas herbaceous species tended towards phylogenetic overdispersion. We also found phylogenetic clustering in two edaphic environments (sedimentary and crystalline) in contrast to phylogenetic overdispersion in the third (inselberg). We conclude that while niche conservatism is evident in phylogenetic clustering in the Caatinga, this is not a universal pattern likely due to heterogeneity in the degree of realized environmental filtering across edaphic environments. Thus, SDTF, in spite of a strong shared environmental filter, are potentially heterogeneous in phylogenetic structuring. Our results support the need for scientifically informed conservation strategies in the Caatinga and other SDTF regions that have not previously been prioritized for conservation in order to take into account this heterogeneity.     

A Role for p38 Stress-Activated Protein Kinase in Regulation of Cell Growth via TORC1

The target of rapamycin (TOR) complex 1 (TORC1) signaling pathway is a critical regulator of translation and cell growth. To identify novel components of this pathway, we performed a kinome-wide RNA interference (RNAi) screen in Drosophila melanogaster S2 cells. RNAi targeting components of the p38 stress-activated kinase cascade prevented the cell size increase elicited by depletion of the TOR negative regulator TSC2. In mammalian and Drosophila tissue culture, as well as in Drosophila ovaries ex vivo, p38-activating stresses, such as H₂O₂ and anisomycin, were able to activate TORC1. This stress-induced TORC1 activation could be blocked by RNAi against mitogen-activated protein kinase kinase 3 and 6 (MKK3/6) or by the overexpression of dominant negative Rags. Interestingly, p38 was also required for the activation of TORC1 in response to amino acids and growth factors. Genetic ablation either of p38b or licorne, its upstream kinase, resulted in small flies consisting of small cells. Mutants with mutations in licorne or p38b are nutrition sensitive; low-nutrient food accentuates the small-organism phenotypes, as well as the partial lethality of the p38b null allele. These data suggest that p38 is an important positive regulator of TORC1 in both mammalian and Drosophila systems in response to certain stresses and growth factors.The target of rapamycin, TOR, is a highly conserved serine/threonine kinase that is a critical regulator of cell growth. It is a core component of two signaling complexes, TORC1 and TORC2 (60, 74). TORC1 is defined by the presence of Raptor in the complex, while TORC2 contains Rictor. Rictor and Raptor are mutually exclusive. Activation of the TORC1 pathway leads to increased protein translation, increased cell size, and increased proliferation, making this pathway an important target for emerging cancer therapies. Rapamycin is an inhibitor of TORC1 that is commonly used as an immunosuppressant following kidney transplantation (51). At least three analogs of rapamycin are currently being tested in solid and hematological tumors and have shown some promising results (21).The TORC1 pathway responds to numerous inputs, sensing both the desirability of and the capacity for growth. Many of these pathways control TORC1 signaling through phosphorylation of the tuberous sclerosis protein TSC2. TSC2 associates with TSC1 to form a heterodimeric GTPase-activating protein complex (GAP) that inactivates the small GTPase Rheb (24, 29, 67). While the exact molecular mechanism remains a topic of debate, activation of Rheb promotes the kinase activity of TORC1 (24, 29, 67). Rheb is required for the activation of TORC1 in response to both amino acids and growth factors (55, 62). In Drosophila melanogaster, mutation of either TOR or Rheb inhibits growth, leading to reduced body size and reduced cell size in mutant clones (42, 64). Mutation of either TSC1 or TSC2 has the predicted opposite effect, as tissue deficient for either of these proteins overgrows and contains large cells (49, 66).TORC1 is activated via the phosphatidylinositol 3′ kinase (PI3′K) pathway by growth-promoting mitogens, such as insulin and growth factors. Drosophila mutants with mutations of PI3′K pathway components have size phenotypes similar to those of the TOR and Rheb mutants (71). In mammalian cells, the PI3′K-mediated activation of TORC1 occurs at least in part through the phosphorylation of TSC2 by the PI3′K target AKT (30, 50). Interestingly, mutation of these residues in Drosophila has no impact on TSC2 function in vivo, suggesting that there may be other mechanisms through which PI3′K can activate Drosophila TOR (20). Recent work has suggested that the proline-rich AKT substrate PRAS40 may provide part of this link (23, 59, 69, 70). In addition, signaling through RAS activates extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK), which can phosphorylate TSC2 and Raptor to activate TORC1 (13, 40, 56). There are also likely to be additional mechanisms through which growth factors activate Drosophila TOR that have not yet been identified.TORC1 activity is also controlled by the intracellular building blocks necessary to support cellular growth. The energy-sensing AMP-activated protein kinase (AMPK) pathway relays information about the energy status of the cell to TORC1 by phosphorylating TSC2. Unlike the inactivating phosphorylation of TSC2 by Akt, phosphorylation of TSC2 by AMPK promotes the GAP activity of the TSC complex (31). AMPK also phosphorylates Raptor, leading to decreased TORC1 activity (28). Thus, when energy levels are low, active AMPK inhibits TORC1.Amino acids also activate the TORC1 pathway, through a mechanism that requires Rheb, as well as the type III PI3′K VPS34 and the serine/threonine kinase mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3) (11, 22, 43). TORC1 thereby integrates information about the availability of amino acids and the amount of energy available for growth with growth factor signaling. Given its ancient function in adapting growth rates to environmental conditions, it is likely that TOR responds to a variety of stimuli, suggesting that many TOR control mechanisms remain to be uncovered. The Rag family of Ras-related small GTPases has recently been identified as a key component of the amino acid-sensing pathway, acting in parallel to Rheb (34, 58). Rag GTPases form heterodimers; RagA or RagB interacts with RagC or RagD. RagA and RagB are active when GTP bound, while RagC and RagD are active when bound to GDP (34, 58). Activation of the Rags by amino acids results in TOR relocalization to Rab7-containing vesicles (58). While the function of these vesicles in TORC1 signaling remains unclear, this relocalization is associated with increased TORC1 activity.TORC1 controls cell growth and translation through the phosphorylation and activation of components of the translational machinery, such as S6 kinase (S6K) and 4EBP1, an inhibitor of eukaryotic translation initiation factor 4E (eIF4E) activity (reviewed in reference 27). S6K phosphorylates the S6 ribosomal subunit, thereby increasing translation. Mice deficient for S6K1 are small and have small pancreatic beta cells and a correspondingly low level of circulating insulin (45). Mutation of the phosphorylation sites on S6 results in a similar phenotype, with small beta cells and fibroblasts (57). In Drosophila, mutation of S6K again reduces both cell and organism size (42), as does the overexpression of 4EBP (41).Interestingly, while mutation of the TORC1 pathway in mammalian cells reduces cell size by 10 to 15%, ablation of core TORC1 pathway components in Drosophila cells can affect cell size by up to 40% (73). In an attempt to identify novel components of the TORC1 pathway, we undertook an RNA interference (RNAi)-based screen of Drosophila S2 cells. We reasoned that the extreme size phenotypes observed in Drosophila cells upon TORC1 manipulations would facilitate the identification of modulators. In order to increase the likelihood of isolating novel regulators of TOR, we uncoupled TOR activity from many of its known nutritional controls by depleting TSC2 and screened for double-stranded RNAs (dsRNAs) that could reverse the cell size increase elicited by loss of TSC2. Depletion of multiple components of the p38 pathway was found to revert the TSC2 RNAi-induced cell size increase. Furthermore, activation of p38 is necessary and sufficient for the activation of TOR. Strikingly, mutation of components of the stress-activated p38 pathway in Drosophila has a similar phenotype to mutations in the TOR and insulin signaling pathway: a cell-autonomous cell size decrease, reduced body size, and a sensitization to the effects of nutritional stress.