Granule-bound starch synthase: structure, function, and phylogenetic utility

Interest in the use of low-copy nuclear genes for phylogenetic analyses of plants has grown rapidly, because highly repetitive genes such as those commonly used are limited in number. Furthermore, because low- copy genes are subject to different evolutionary processes than are plastid genes or highly repetitive nuclear markers, they provide a valuable source of independent phylogenetic evidence. The gene for granule-bound starch synthase (GBSSI or waxy) exists in a single copy in nearly all plants examined so far. Our study of GBSSI had three parts: (1) Amino acid sequences were compared across a broad taxonomic range, including grasses, four dicotyledons, and the microbial homologs of GBSSI. Inferred structural information was used to aid in the alignment of these very divergent sequences. The informed alignments highlight amino acids that are conserved across all sequences, and demonstrate that structural motifs can be highly conserved in spite of marked divergence in amino acid sequence. (2) Maximum-likelihood (ML) analyses were used to examine exon sequence evolution throughout grasses. Differences in probabilities among substitution types and marked among-site rate variation contributed to the observed pattern of variation. Of the parameters examined in our set of likelihood models, the inclusion of among-site rate variation following a gamma distribution caused the greatest improvement in likelihood score. (3) We performed cladistic parsimony analyses of GBSSI sequences throughout grasses, within tribes, and within genera to examine the phylogenetic utility of the gene. Introns provide useful information among very closely related species, but quickly become difficult to align among more divergent taxa. Exons are variable enough to provide extensive resolution within the family, but with low bootstrap support. The combined results of amino acid sequence comparisons, maximum-likelihood analyses, and phylogenetic studies underscore factors that might affect phylogenetic reconstruction. In this case, accommodation of the variable rate of evolution among sites might be the first step in maximizing the phylogenetic utility of GBSSI.      

Archaeal overdominance close to life-limiting conditions in geothermally influenced hypersaline lakes at the Danakil Depression,Ethiopia

The Dallol protovolcanic area on the Danakil Depression (Afar region, Ethiopia) exhibits unique hydrothermal manifestations in hypersaline context, yielding varied polyextreme physicochemical conditions. Previous studies identified a wide archaeal diversity in less extreme brines but failed to identify microorganisms thriving in either high-chaotropicity, low-water-activity brines or hyperacidic-hypersaline Na-Fe-rich brines. Recently, we accessed several small lakes under intense degassing activity adjacent to the Round Mountain, west to the Dallol dome [Western Canyon Lakes (WCL); WCL1-5]. They exhibited intermediate parameter combinations (pH ~ 5, 34%–41% (weight/volume) NaCl-dominated salts with relatively high levels of chaotropic Mg-Ca salts) that should allow to better constrain life limits. These lakes were overwhelmingly dominated by Archaea, encompassing up to 99% of prokaryotic 16S rRNA gene amplicon sequences in metabarcoding studies. The majority belonged to Halobacteriota and Nanohaloarchaeota, the latter representing up to half of prokaryotic sequences. Optical and epifluorescence microscopy showed active cells in natural samples and diverse morphotypes in enrichment cultures. Scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy revealed tiny cells (200–300 nm diameter) epibiotically associated with somewhat larger cells (0.6–1 μm) but also the presence of silica-dominated precipitates of similar size and shape, highlighting the difficulty of distinguishing microbes from mineral biomorphs in this kind of low-biomass systems.     

Hypoxia and vitamin D differently contribute to leptin and dickkopf-related protein 2 production in human osteoarthritic subchondral bone osteoblasts

Introduction

Bone remodelling and increased subchondral densification are important in osteoarthritis (OA). Modifications of bone vascularization parameters, which lead to ischemic episodes associated with hypoxic conditions, have been suspected in OA. Among several factors potentially involved, leptin and dickkopf-related protein 2 (DKK2) are good candidates because they are upregulated in OA osteoblasts (Obs). Therefore, in the present study, we investigated the hypothesis that hypoxia may drive the expression of leptin and DKK2 in OA Obs.

Methods

Obs from the sclerotic portion of OA tibial plateaus were cultured under either 20% or 2% oxygen tension in the presence or not of 50 nM 1,25-dihydroxyvitamin D₃ (VitD₃). The expression of leptin, osteocalcin, DKK2, hypoxia-inducible factor 1α (Hif-1α) and Hif-2α was measured by real-time polymerase chain reaction and leptin production was measured by enzyme-linked immunosorbent assay (ELISA). The expression of Hif-1α, Hif-2α, leptin and DKK2 was reduced using silencing RNAs (siRNAs). The signalling pathway of hypoxia-induced leptin was investigated by Western blot analysis and with mitogen-activated protein kinase (MAPK) inhibitors.

Results

The expression of leptin and DKK2 in Obs was stimulated 7-fold and 1.8-fold, respectively (P <0.05) under hypoxia. Interestingly, whereas VitD₃ stimulated leptin and DKK2 expression 2- and 4.2-fold, respectively, under normoxia, it stimulated their expression by 28- and 6.2-fold, respectively, under hypoxia (P <0.05). The hypoxia-induced leptin production was confirmed by ELISA, particularly in the presence of VitD₃ (P <0.02). Compared to Obs incubated in the presence of scramble siRNAs, siHif-2α inhibited VitD₃-stimulated leptin mRNA and protein levels by 70% (P =0.004) and 60% (P <0.02), respectively, whereas it failed to significantly alter the expression of DKK2. siHif-1α has no effect on these genes. Immunoblot analysis showed that VitD₃ greatly stabilized Hif-2α under hypoxic conditions. The increase in leptin expression under hypoxia was also regulated, by p38 MAPK (P <0.03) and phosphoinositide 3-kinase (P <0.05). We found that the expression of leptin and DKK2 were not related to each other under hypoxia.

Conclusions

Hypoxic conditions via Hif-2 regulation trigger Obs to produce leptin, particularly under VitD₃ stimulation, whereas DKK2 is regulated mainly by VitD₃ rather than hypoxia.     

Early calcium handling imbalance in pressure overload-induced heart failure with nearly normal left ventricular ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is a common clinical syndrome associated with high morbidity and mortality. Therapeutic options are limited due to a lack of knowledge of the pathology and its evolution. We investigated the cellular phenotype and Ca²⁺ handling in hearts recapitulating HFpEF criteria. HFpEF was induced in a portion of male Wistar rats four weeks after abdominal aortic banding. These animals had nearly normal ejection fraction and presented elevated blood pressure, lung congestion, concentric hypertrophy, increased LV mass, wall stiffness, impaired active relaxation and passive filling of the left ventricle, enlarged left atrium, and cardiomyocyte hypertrophy. Left ventricular cell contraction was stronger and the Ca²⁺ transient larger. Ca²⁺ cycling was modified with a RyR2 mediated Ca²⁺ leak from the sarcoplasmic reticulum and impaired Ca²⁺ extrusion through the Sodium/Calcium exchanger (NCX), which promoted an increase in diastolic Ca²⁺. The Sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA2a) and NCX protein levels were unchanged. The phospholamban (PLN) to SERCA2a ratio was augmented in favor of an inhibitory effect on the SERCA2a activity. Conversely, PLN phosphorylation at the calmodulin-dependent kinase II (CaMKII)-specific site (PLN-Thr17), which promotes SERCA2A activity, was increased as well, suggesting an adaptive compensation of Ca²⁺ cycling. Altogether our findings show that cardiac remodeling in hearts with a HFpEF status differs from that known for heart failure with reduced ejection fraction. These data also underscore the interdependence between systolic and diastolic “adaptations” of Ca²⁺ cycling with complex compensative interactions between Ca²⁺ handling partner and regulatory proteins.     

Gradient of distribution in Europe of the major CF mutation and of its associated haplotype

Summary In this collaborative European study, a total of 4871 cystic fibrosis (CF) chromosomes and 3539 normal chromosomes have been characterized for the haplotypes defined by the 2 extragenic polymorphic sequences revealed by XV2c and KM19. The association between one of these haplotypes (B haplotype) and the most frequent CF mutation, ΔF508, suggests for the latter a single origin and a subsequent diffusion according to a South East-North West gradient. The linkage disequilibrium data between CF and the B haplotype in different European populations are compatible with a relatively more recent appearance of the mutation in Northern Europe whereas in Southern Europe a longer history of the same mutation would have allowed time for recombination with other haplotypes. This model is also compatible with a selective advantage of carriers but does not account for (1) the excess of B haplotypes observed among both normal and non-ΔF508 CF chromosomes; (2) the correlation between the B haplotype and the severity of the phenotypic effect caused by CF mutations, as measured by pancreatic insufficiency and meconium ileus.     

Study of O-glycan sialylation in C6 cultured glioma cells: regulation of a beta-galactoside alpha 2,3 sialyltransferase activity by Ca2+/calmodulin antagonists and phosphatase inhibitors.

We have demonstrated that the alpha 2,3 sialyltransferase (alpha 2,3 ST) from C6 cultured glioma cells was inhibited in vivo by W-7 and related Ca2+/Calmodulin (Ca/CaM) antagonists while protein kinase C effectors had no effect. Dephosphorylation of alpha 2,3 ST by the wide specificity alkaline phosphatase led to inactivation indicating that the enzyme is phosphorylated. The serine/threonine protein phosphatase inhibitors okadaic acid and Calyculin A led also to an inhibition of alpha 2,3 ST activity. In addition, Ca/CaM antagonists and phosphatase inhibitors led both to an inhibition of a alpha 2,3 sialoglycoprotein from C6 glioma cells as demonstrated with lectin affinity blotting. A concerted regulatory mechanism with phosphorylation/dephosphorylation of alpha 2,3 ST is then postulated.