Organic act rejected in the cordillera: Dialectics of a continuing fourth world autonomy movement in the Philippines

David Hyndman is Professor in the Department of Anthropology and Sociology, University of Queensland, Australia     

Evidence for a novel GTPase priming step in the SRP protein targeting pathway.

Protein targeting by the signal recognition particle (SRP) pathway requires the interaction of two homologous GTPases that reciprocally regulate each other's GTPase activity, the SRP signal peptide- binding subunit (SRP54) and the SRP receptor alpha-subunit (SRalpha). The GTPase domain of both proteins abuts a unique 'N domain' that appears to facilitate external ligand binding. To examine the relationship between the unusual regulation and unique architecture of the SRP pathway GTPases, we mutated an invariant glycine in Escherichia coli SRP54 and SRalpha orthologs ('Ffh' and 'FtsY', respectively) that resides at the N-GTPase domain interface. A G257A mutation in Ffh produced a lethal phenotype. The mutation did not significantly affect Ffh function, but severely reduced interaction with FtsY. Likewise, mutation of FtsY Gly455 produced growth defects and inhibited interaction with Ffh. The data suggest that Ffh and FtsY interact only in a 'primed' conformation which requires interdomain communication. Based on these results, we propose that the distinctive features of the SRP pathway GTPases evolved to ensure that SRP and the SR engage external ligands before interacting with each other.     

Spatial and temporal changes in microbial community structure associated with recharge-influenced chemical gradients in a contaminated aquifer

In a contaminated water-table aquifer, we related microbial community structure on aquifer sediments to gradients in 24 geochemical and contaminant variables at five depths, under three recharge conditions. Community amplified ribsosomal DNA restriction analysis (ARDRA) using universal 16S rDNA primers and denaturing gradient gel electrophoresis (DGGE) using bacterial 16S rDNA primers indicated: (i). communities in the anoxic, contaminated central zone were similar regardless of recharge; (ii). after recharge, communities at greatest depth were similar to those in uncontaminated zones; and (iii). after extended lack of recharge, communities at upper and lower aquifer margins differed from communities at the same depths on other dates. General aquifer geochemistry was as important as contaminant or terminal electron accepting process (TEAP) chemistry in discriminant analysis of community groups. The Shannon index of diversity (H) and the evenness index (E), based on DGGE operational taxonomic units (OTUs), were statistically different across community groups and aquifer depths. Archaea or sulphate-reducing bacteria 16S rRNA abundance was not clearly correlated with TEAP chemistry indicative of methanogenesis or sulphate reduction. Eukarya rRNA abundance varied by depth and date from 0 to 13% of the microbial community. This contaminated aquifer is a dynamic ecosystem, with complex interactions between physical, chemical and biotic components, which should be considered in the interpretation of aquifer geochemistry and in the development of conceptual or predictive models for natural attenuation or remediation.     

Physiological basis for conservation of the signal recognition particle targeting pathway in Escherichia coli

The Escherichia coli signal recognition particle (SRP) is a ribonucleoprotein complex that targets nascent inner membrane proteins (IMPs) to transport sites in the inner membrane (IM). Since SRP depletion only partially inhibits IMP insertion under some growth conditions, however, it is not clear why the particle is absolutely essential for viability. Insights into this question emerged from experiments in which we analyzed the physiological consequences of reducing the intracellular concentration of SRP below the wild-type level. We found that even moderate SRP deficiencies that have little effect on cell growth led to the induction of a heat shock response. Genetic manipulations that suppress the heat shock response were lethal in SRP-deficient cells, indicating that the elevated synthesis of heat shock proteins plays an important role in maintaining cell viability. Although it is conceivable that the heat shock response serves to increase the capacity of cells to target IMPs via chaperone-based mechanisms, SRP-deficient cells did not show an increased dependence on either GroEL or DnaK. By contrast, the heat shock-regulated proteases Lon and ClpQ became essential for viability when SRP levels were reduced. These results suggest that the heat shock response protects SRP-deficient cells by increasing their capacity to degrade mislocalized IMPs. Consistent with this notion, a model IMP that was mislocalized in the cytoplasm as the result of SRP depletion appeared to be more stable in a Deltalon DeltaclpQ strain than in control cells. Taken together, the data provide direct evidence that SRP is essential in E. coli and possibly conserved throughout prokaryotic evolution as well partly because efficient IMP targeting prevents a toxic accumulation of aggregated proteins in the cytoplasm.     

Differential role of CENP-A in the segregation of holocentric C. elegans chromosomes during meiosis and mitosis

Two distinct chromosome architectures are prevalent among eukaryotes: monocentric, in which localized centromeres restrict kinetochore assembly to a single chromosomal site, and holocentric, in which diffuse kinetochores form along the entire chromosome length. During mitosis, both chromosome types use specialized chromatin, containing the histone H3 variant CENP-A, to direct kinetochore assembly. For the segregation of recombined homologous chromosomes during meiosis, monocentricity is thought to be crucial for limiting spindle-based forces to one side of a crossover and to prevent recombined chromatids from being simultaneously pulled towards both spindle poles. The mechanisms that allow holocentric chromosomes to avert this fate remain uncharacterized. Here, we show that markedly different mechanisms segregate holocentric chromosomes during meiosis and mitosis in the nematode Caenorhabditis elegans. Immediately prior to oocyte meiotic segregation, outer-kinetochore proteins were recruited to cup-like structures on the chromosome surface via a mechanism that is independent of CENP-A. In striking contrast to mitosis, both oocyte meiotic divisions proceeded normally following depletion of either CENP-A or the closely associated centromeric protein CENP-C. These findings highlight a pronounced difference between the segregation of holocentric chromosomes during meiosis and mitosis and demonstrate the potential to uncouple assembly of outer-kinetochore proteins from CENP-A chromatin.     

Short-term low-salinity tolerance by the longhorn sculpin, Myoxocephalus octodecimspinosus

The bottom-dwelling, longhorn sculpin, Myoxocephalus octodecimspinosus, is traditionally viewed as a stenohaline marine fish, but fishermen have described finding this sculpin in estuaries during high tide. Little is known about the salinity tolerance of the longhorn sculpin; thus, the purposes of these experiments were to explore the effects of low environmental salinity on ion transporter expression and distribution in the longhorn sculpin gill. Longhorn sculpin were acclimated to either 100% seawater (SW, sham), 20% SW, or 10% SW for 24 or 72 hr. Plasma osmolality, sodium, potassium, and chloride concentrations were not different between the 20 and 100% treatments; however, they were 20-25% lower with exposure to 10% SW at 24 and 72 hr. In the teleost gill, regulation of Na(+), K(+)-ATPase (NKA), Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), and the chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR) are necessary for ion homeostasis. We immunolocalized these proteins to the mitochondrion-rich cell of the gill and determined that acclimation to low salinity does not affect their localization. Also, there was not a downregulation of gill NKA, NKCC1, and CFTR mRNA or protein during acclimation to low salinities. Collectively, these results suggest that down to 20% SW longhorn sculpin are capable of completely regulating ion levels over a 72-hr period, whereas 10% SW exposure results in a significant loss of ions and no change in ion transporter density or localization in the gill. We conclude that longhorn sculpin can tolerate low-salinity environments for days but, because they cannot regulate ion transporter density, they are unable to tolerate low salinity for longer periods or enter freshwater (FW). The genus Myoxocephalus has three FW species, making this group an excellent model to test evolutionary and physiological mechanisms that allow teleosts to invade new low salinities successfully.     

Dynamin activates NO production in rat renal inner medullary collecting ducts via protein-protein interaction with NOS1

We hypothesized that nitric oxide synthase (NOS) isoforms may be regulated by dynamin (DNM) in the inner medullary collecting duct (IMCD). The aims of this study were to determine which DNM isoforms (DNM1, DNM2, DNM3) are expressed in renal IMCDs, whether DNM interacts with NOS, whether a high-salt diet alters the interaction of DNM and NOS, and whether DNM activates NO production. DNM2 and DNM3 are highly expressed in the rat IMCD, while DNM1 is localized outside of the IMCD. We found that DNM1 interacts with NOS1α, NOS1β, and NOS3 in the inner medulla of male Sprague-Dawley rats on a 0.4% salt diet. DNM2 interacts with NOS1α, while DNM3 interacts with both NOS1α and NOS1β. DNM2 and DNM3 do not interact with NOS3 in the rat inner medulla. We did not observe any change in the DNM/NOS interactions with rats on a 4% salt diet after 7 days. Furthermore, NOS1α interacts with DNM2 in mIMCD3 and COS7 cells transfected with NOS1α and DNM2-GFP constructs and the NOS1 reductase domain is necessary for the interaction. Finally, COS7 cells expressing NOS1α or NOS1α/DNM2-GFP had significantly higher nitrite production compared with DNM2-GFP only. Nitrite production was blocked by the DNM inhibitor dynasore or the dominant negative DNM2K44A. Ionomycin stimulation further increased nitrite production in the NOS1α/DNM2-GFP cells compared with NOS1α only. In conclusion, DNM and NOS1 interact in the rat renal IMCD and this interaction leads to increased NO production, which may influence NO production in the renal medulla.     

Origins of the Extracellular Glutamate Released During Total Metabolic Blockade in the Immature Retina

Abstract: Previous studies have shown that complete blockade of metabolism in embryonic chick retina causes a time-dependent increase in the release of glutamate into the extracellular space. The present study examined the cellular source of this glutamate, i.e., neuronal and/or glial. Pure cultures of retinal neurons or glia were labeled for 10 min at 37°C with [³H]acetate. Retinal glia, but not retinal neurons, were found to selectively and preferentially metabolize acetate, thus producing ³H-labeled amino acids in the glial compartment. This finding provides direct evidence to substantiate findings from several other laboratories that have indirectly determined the preferential metabolism of acetate by glia by using mixed neuronal/glial populations. To study the cellular source of glutamate released during total metabolic blockade, whole retina were prelabeled with [³H]acetate plus [U-¹⁴C]glucose (to label the neuronal compartment). Total metabolic blockade was instituted with a combination of iodoacetate (IOA) plus KCN, and the release of glutamate into the medium was followed at 5, 15, and 30 min. During total energy blockade, net extracellular glutamate was not elevated at 5 min [0.17 ± 0.02 vs. 0.12 ± 0.01 µM for treated vs. control retina (means ± SEM), respectively], but was increased significantly at 15 (1.2 ± 0.26 µM) and 30 min (2.6 ± 0.22 µM). Total [³H]glutamate in the medium during IOA/KCN treatment was unchanged at 5 min, but was increased 1.5- and threefold above basal levels at 15 and 30 min, respectively. During the time when extracellular glutamate increased, the specific activity of [³H]glutamate remained fairly constant, 731 ± 134 and 517 ± 82 dpm/nmol (means ± SEM) at 15 and 30 min, respectively. In contrast, ¹⁴C-labeled glutamate in the medium did not increase during IOA/KCN treatment and paralleled basal levels. Thus, the specific activity of ¹⁴C-labeled extracellular glutamate decreased from 309 ± 87 dpm/nmol at 15 min to 42 ± 8 dpm/nmol at 30 min. Prior loading of the tissue with 0.5 mM trans-pyrrolidine-2,4-dicarboxylate (t-PDC), a glutamate transport inhibitor, blocked 57% of the glutamate released at 30 min of IOA/KCN exposure, suggesting that reversal of an Na⁺-dependent glutamate transporter was a key contributor to the appearance of extracellular glutamate during energy deprivation. The increase in extracellular [³H]glutamate, constancy of the specific activity of extracellular [³H]glutamate, decrease in the specific activity of extracellular [¹⁴C]glutamate, and attenuation of release by prior loading with t-PDC indicate that glial pools of glutamate released via reversal of the transporter contribute significantly to the rise in extracellular glutamate after metabolic inhibition in this preparation.