The A-kinase Anchoring Protein GSKIP Regulates GSK3β Activity and Controls Palatal Shelf Fusion in Mice

A-kinase anchoring proteins (AKAPs) represent a family of structurally diverse proteins, all of which bind PKA. A member of this family is glycogen synthase kinase 3β (GSK3β) interaction protein (GSKIP). GSKIP interacts with PKA and also directly interacts with GSK3β. The physiological function of the GSKIP protein in vivo is unknown. We developed and characterized a conditional knock-out mouse model and found that GSKIP deficiency caused lethality at birth. Embryos obtained through Caesarean section at embryonic day 18.5 were cyanotic, suffered from respiratory distress, and failed to initiate breathing properly. Additionally, all GSKIP-deficient embryos showed an incomplete closure of the palatal shelves accompanied by a delay in ossification along the fusion area of secondary palatal bones. On the molecular level, GSKIP deficiency resulted in decreased phosphorylation of GSK3β at Ser-9 starting early in development (embryonic day 10.5), leading to enhanced GSK3β activity. At embryonic day 18.5, GSK3β activity decreased to levels close to that of wild type. Our findings reveal a novel, crucial role for GSKIP in the coordination of GSK3β signaling in palatal shelf fusion.     

Noninvasive determination of local pulse wave velocity and wave intensity: changes with age and gender in the carotid and femoral arteries of healthy human

We recently introduced noninvasive methods to assess local pulse wave velocity (PWV) and wave intensity ((n)dI) in arteries based on measurements of flow velocity (U) and diameter (D). Although the methods were validated in an experimental setting, clinical application remains lacking. The aim of this study was therefore to investigate the effect of age and gender on PWV and (n)dI in the carotid and femoral arteries of an existing population. We measured D and U in the carotid and femoral arteries of 1,774 healthy subjects aged 35-55 yr, a subgroup of the Asklepios population. With the use of the lnDU-loop method, we calculated local PWV, which was used to determine arterial distensibility ((n)Ds). We then used the new algorithm to determine maximum forward and backward wave intensities ((n)dI(+max) and (n)dI(-min), respectively) and the reflection index ((n)RI). On average, PWV was higher, and (n)Ds was lower in the femoral than at the carotid arteries. At the carotid artery, PWV increased with age, but (n)Ds, (n)dI(+max), and (n)dI(-min) decreased; (n)RI did not change with age. At the femoral artery, PWV was higher, and (n)Ds was lower in male, but all parameters did not change significantly with age in both women and men. We conclude that the carotid artery is more affected by the aging process than the femoral artery, even in healthy subjects. The new techniques provide mechanical and hemodynamic parameters, requiring only D and U measurements, both of which can be acquired using ultrasound equipment widely available today, hence their advantage for potential use in the clinical setting.     

16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales"

For simultaneous identification of members of the betaproteobacterial order "Rhodocyclales" in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the "Rhodocyclales." The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent "Rhodocyclales" diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed "Rhodocyclales"-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of "Rhodocyclales" populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.     

Regulation of ion transport by pH and [HCO3-] in isolated gills of the crab Neohelice (Chasmagnathus) granulata

Posterior isolated gills of Neohelice (Chasmagnathus) granulatus were symmetrically perfused with hemolymph-like saline of varying [HCO3-] and pH. Elevating [HCO3-] in the saline from 2.5 to 12.5 mmol/l (pH 7.75 in both cases) induced a significant increase in the transepithelial potential difference (Vte), a measure of ion transport. The elevation in [HCO3-] also induced a switch from acid secretion (-43.7 +/- 22.5 microequiv.kg(-1).h(-1)) in controls to base secretion (84.7 +/- 14.4 microequiv.kg(-1).h(-1)). The HCO3(-)-induced Vte increase was inhibited by basolateral acetazolamide (200 micromol/l), amiloride (1 mmol/l), and ouabain (5 mmol/l) but not by bafilomycin (100 nmol/l). The Vte response to HCO3(-) did not take place in Cl(-)-free conditions; however, it was unaffected by apical SITS (2 mmol/l) or DIDS (1 mmol/l). A decrease in pH from 7.75 to 7.45 pH units in the perfusate also induced a significant increase in Vte, which was matched by a net increase in acid secretion of 67.8 +/- 18.4 microequiv kg(-1) h(-1). This stimulation was sensitive to basolateral acetazolamide, bafilomycin, DIDS, and Na+-free conditions, but it still took place in Cl(-)-free saline. Therefore, the cellular response to low pH is different from the HCO3(-)-stimulated response. We also report V-H+-ATPase- and Na+-K+-ATPase-like immunoreactivity in gill sections for the first time in this crab. Our results suggest that carbonic anhydrase (CA), basolateral Na+/H+ exchangers and Na+-K+-ATPase and apical anion exchangers participate in the HCO3(-)-stimulated response, while CA, apical V-H+-ATPase and basolateral HCO3(-)-dependent cotransporters mediate the response to low pH.     

<Superscript>13</Superscript>C labeling experiments at metabolic nonstationary conditions: An exploratory study

Background  

Stimulus Response Experiments to unravel the regulatory properties of metabolic networks are becoming more and more popular. However, their ability to determine enzyme kinetic parameters has proven to be limited with the presently available data. In metabolic flux analysis, the use of ¹³C labeled substrates together with isotopomer modeling solved the problem of underdetermined networks and increased the accuracy of flux estimations significantly.     

Plasmodium falciparum dolichol phosphate mannose synthase represents a novel clade

Dolichol phosphate mannose synthase (DPM) catalyzes the reaction between dolichol phosphate (Dol-P) and guanosine diphosphate mannose (GDP-Man) to form dolichol-phosphate-mannose (Dol-P-Man). This molecule acts as mannose donor for N-glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis. The Plasmodium falciparum DPM1 (Pfdpm1) possesses a single predicted transmembrane region near the N-, but not the C-terminus. Here we show that the cloned Pfdpm1 gene failed to complement a Saccharomyces cerevisiae mutant indicating that the parasite gene does not belong to the baker’s yeast group, as was previously assumed. Furthermore, Pfdpm1 was unable to complement a mouse mutant deficient in DPM but efficiently complements the Schizosaccharomyces pombe fission yeast mutant, indicating a difference between fission yeast and mammalian DPM genes. Therefore, we reanalyzed the hydrophobicity scales of all known DPMs and consequently reclassify the DPM clade into six major novel subgroups. Furthermore, we show that Pfdpm1 represents a unique enzyme among these subgroups.