Role of the cgtA gene function in DNA replication of extrachromosomal elements in Escherichia coli

The cgtA gene codes for a common GTP-binding protein whose homologues were found in all prokaryotic and eukaryotic organisms investigated so far. Although cgtA is an essential gene in most bacterial species, its precise functions in the regulation of cellular processes are largely unknown. In Escherichia coli, dysfunction or overexpression of the cgtA gene causes problems in various chromosomal functions, like synchronization of DNA replication initiation and partitioning of daughter chromosomes after a replication round. It is not know how the cgtA gene product regulates these processes. Here we investigated effects of cgtA dysfunction on replication of plasmid and phage replicons. We found that replication of some plasmids (e.g., ColE1-like) is not affected in the cgtA mutant. On the other hand, dysfunction of the cgtA gene caused a strong inhibition of lambda plasmid DNA replication. Bacteriophage lambda development was severely impaired in the cgtA mutant. Replication of other plasmid replicons (derivatives of F, R1, R6K, and RK2) was influenced by the cgtA mutation moderately. It seems that DNA synthesis per se is not affected by CgtA, and that this protein might control replication initiation indirectly, by regulation of function(s) or production of one or more replication factors. In fact, we found that level of the host-encoded replication protein DnaA is significantly decreased in the cgtA mutant. This indicates that CgtA is involved in the regulation of dnaA gene expression.     

Methanogenic potential of Arctic and Antarctic subglacial environments with contrasting organic carbon sources

Subglacial environments are largely anoxic, contain organic carbon (OC) overridden by glacier ice during periods of advance, and harbour active microbial communities. This creates favourable conditions for OC degradation via methanogenesis. It has been hypothesized that OC beneath ice sheets is converted to methane (CH₄) and may be released to the atmosphere during retreat. However, there are limited data available to support this contention. Here, we present new data on the abundance, diversity and activity of methanogenic archaea and the amount and character of OC in subglacial sediments from Arctic and Antarctic glacial systems based on different substrate types. We employed long‐term laboratory incubations to quantify the CH₄ production potential in different subglacial settings. Significant numbers of methanogens (up to 7 × 10⁴ cells g^?1) were detected in the samples and clones of Methanomicrobiales and Methanosarcinales were identified in clone libraries. Long lag periods (up to >200 days) were observed before significant CH₄ concentrations were measured. We report order of magnitude differences in rates of CH₄ production (10¹–10⁵ fmol g^?1 d^?1) in different subglacial sediments, reflecting contrasts in the origin of the sediment and the OC character. Hence, we predict that contrasting rates of CH₄ production are likely to occur beneath glaciers and ice sheets that overran different types of substrate. We subsequently estimated the potential for CH₄ production beneath the Laurentide/Inuitian/Cordilleran and Fennoscandian Ice Sheets during a typical 85 ka Quaternary glacial/interglacial cycle. CH₄ production from lacustrine‐derived OC is likely to be an order of magnitude higher (~6.3–27 Pg C) than that from overridden soils (~0.55–0.68 Pg C), possibly due to a difference in lability between lacustrine and soil OC. While representing a fraction of the entire OC pool (~418–610 Pg C), this finding highlights the importance of considering the character of different OC pools when calculating subglacial CH₄ production.     

Functional Loss of Semaphorin 3C and/or Semaphorin 3D and Their Epistatic Interaction with Ret Are Critical to Hirschsprung Disease Liability

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.     

Cytosolic phospholipase A2alpha activation induced by S1P is mediated by the S1P3 receptor in lung epithelial cells

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) activation is a regulatory step in the control of arachidonic acid (AA) liberation for eicosanoid formation. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator involved in the regulation of many important proinflammatory processes and has been found in the airways of asthmatic subjects. We investigated the mechanism of S1P-induced AA release and determined the involvement of cPLA(2)alpha in these events in A549 human lung epithelial cells. S1P induced AA release rapidly within 5 min in a dose- and time-dependent manner. S1P-induced AA release was inhibited by the cPLA(2)alpha inhibitors methyl arachidonyl fluorophosphonate (MAFP) and pyrrolidine derivative, by small interfering RNA-mediated downregulation of cPLA(2)alpha, and by inhibition of S1P-induced calcium flux, suggesting a significant role of cPLA(2)alpha in S1P-mediated AA release. Knockdown of the S1P3 receptor, the major S1P receptor expressed on A549 cells, inhibited S1P-induced calcium flux and AA release. The S1P-induced calcium flux and AA release was associated with sphingosine kinase 1 (Sphk1) expression and activity. Furthermore, Rho-associated kinase, downstream of S1P3, was crucial for S1P-induced cPLA(2)alpha activation. Our data suggest that S1P acting through S1P3, calcium flux, and Rho kinase activates cPLA(2)alpha and releases AA in lung epithelial cells. An understanding of S1P-induced cPLA(2)alpha activation mechanisms in epithelial cells may provide potential targets to control inflammatory processes in the lung.     

Acute impact of blood flow restriction on strength-endurance performance during the bench press exercise

The main goal of the present study was to evaluate the acute effects of blood flow restriction (BFR) at 70% of full arterial occlusion pressure on strength-endurance performance during the bench press exercise. The study included 14 strength-trained male subjects (age = 25.6 ± 4.1 years; body mass = 81.7 ± 10.8 kg; bench press 1 repetition maximum (1RM) = 130.0 ± 22.1 kg), experienced in resistance training (3.9 ± 2.4 years). During the experimental sessions in a randomized crossover design, the subjects performed three sets of the bench press at 80% 1RM performed to failure with two different conditions: without BFR (CON); and with BFR (BFR). Friedman’s test showed significant differences between BFR and CON conditions for the number of repetitions performed (p < 0.001); for peak bar velocity (p < 0.001) and for mean bar velocity (p < 0.001). The pairwise comparisons showed a significant decrease for peak bar velocity and mean bar velocity in individual Set 1 for BFR when compared to CON conditions (p = 0.01 for both). The two-way repeated measures ANOVA showed a significant main effect for the time under tension (p = 0.02). A post-hoc comparisons for the main effect showed a significant increase in time under tension for BFR when compared to CON (p = 0.02). The results of the presented study indicate that BFR used during strength-endurance exercise generally does not decrease the level of endurance performance, while it causes a drop in bar velocity.     

The intracellular distribution of inorganic carbon fixing enzymes does not support the presence of a C4 pathway in the diatom <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis>

Diatoms are unicellular algae and important primary producers. The process of carbon fixation in diatoms is very efficient even though the availability of dissolved CO₂ in sea water is very low. The operation of a carbon concentrating mechanism (CCM) also makes the more abundant bicarbonate accessible for photosynthetic carbon fixation. Diatoms possess carbonic anhydrases as well as metabolic enzymes potentially involved in C4 pathways; however, the question as to whether a C4 pathway plays a general role in diatoms is not yet solved. While genome analyses indicate that the diatom Phaeodactylum tricornutum possesses all the enzymes required to operate a C4 pathway, silencing of the pyruvate orthophosphate dikinase (PPDK) in a genetically transformed cell line does not lead to reduced photosynthetic carbon fixation. In this study, we have determined the intracellular location of all enzymes potentially involved in C4-like carbon fixing pathways in P. tricornutum by expression of the respective proteins fused to green fluorescent protein (GFP), followed by fluorescence microscopy. Furthermore, we compared the results to known pathways and locations of enzymes in higher plants performing C3 or C4 photosynthesis. This approach revealed that the intracellular distribution of the investigated enzymes is quite different from the one observed in higher plants. In particular, the apparent lack of a plastidic decarboxylase in P. tricornutum indicates that this diatom does not perform a C4-like CCM.     

Morphological differences between violets growing at heavy metal polluted and non-polluted sites

Environmental conditions, especially pollution of soils by heavy metals, can severely affect plant morphology, anatomy and reproduction. In present investigation, the morphological variability of forest violets (Viola reichenbachiana and V. riviniana), their degree of colonization by arbuscular mycorrhizal fungi (AMF) and their reproduction properties were studied using samples from ore-bearing areas with soils rich in heavy metals. In these habitats, individuals with typical morphological characters of V. reichenbachiana and V. riviniana were not identified but populations were intermediate between these two species. Three ore-bearing areas with soils of a high content but low extractable amounts of heavy metals (Pb, Zn, Cd) were covered by anthropogenic pine or beech forests, contrary to typical calamine heaps with metallophyte floras. Violets growing on ore-bearing areas were fully fertile, deposited Pb and Zn mainly in roots and had low Cd levels both in shoots and roots. Plants differed in AMF colonization with the degrees varying from nil to moderate. The violets growing in these areas were not affected in their reproduction and can be considered as stabilized introgressive forms or ecotypes of parental species on these soil conditions.     

Phosphorylation of the cyclin-dependent kinase inhibitor p21Cip1 on serine 130 is essential for viral cyclin-mediated bypass of a p21Cip1-imposed G1 arrest

K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.