An andersen-Tawil syndrome mutation in Kir2.1 (V302M) alters the G-loop cytoplasmic K+ conduction pathway

Loss-of-function mutations in the inward rectifier potassium channel, Kir2.1, cause Andersen-Tawil syndrome (ATS-1), an inherited disorder of periodic paralysis and ventricular arrhythmias. Here, we explore the mechanism by which a specific ATS-1 mutation (V302M) alters channel function. Val-302 is located in the G-loop, a structure that is believed to form a flexible barrier for potassium permeation at the apex of the cytoplasmic pore. Consistent with a role in stabilizing the G-loop in an open conformation, we found the V302M mutation specifically renders the channel unable to conduct potassium without altering subunit assembly or attenuating cell surface expression. As predicted by the position of the Val-302 side chain in the crystal structure, amino acid substitution analysis revealed that channel activity and phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivity are profoundly sensitive to alterations in the size, shape, and hydrophobicity of side chains at the Val-302 position. The observations establish that the Val-302 side chain is a critical determinant of potassium conduction through the G-loop. Based on our functional studies and the cytoplasmic domain crystal structure, we suggest that Val-302 may influence PIP2 gating indirectly by translating PIP2 binding to conformational changes in the G-loop pore.     

Hypertension resistance polymorphisms in ROMK (Kir1.1) alter channel function by different mechanisms

The renal outer medullary K(+) (ROMK) channel plays a critical role in renal sodium handling. Recent genome sequencing efforts in the Framingham Heart Study offspring cohort (Ji W, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State MW, Levy D, and Lifton RP. Nat Genet 40: 592-599, 2008) recently revealed an association between suspected loss-of-function polymorphisms in the ROMK channel and resistance to hypertension, suggesting that ROMK activity may also be a determinant of blood pressure control in the general population. Here we examine whether these sequence variants do, in fact, alter ROMK channel function and explore the mechanisms. As assessed by two-microelectrode voltage clamp in Xenopus oocytes, 3/5 of the variants (R193P, H251Y, and T313FS) displayed an almost complete attenuation of whole cell ROMK channel activity. Surface antibody binding measurements of external epitope-tagged channels and analysis of glycosylation-state maturation revealed that these variants prevent channel expression at the plasmalemma, likely as a consequence of retention in the endoplasmic reticulum. The other variants (P166S, R169H) had no obvious effects on the basal channel activity or surface expression but, instead, conferred a gain in regulated-inhibitory gating. As assessed in giant excised patch-clamp studies, apparent phosphotidylinositol 4,5-bisphosphate (PIP(2)) binding affinity of the variants was reduced, causing channels to be more susceptible to inhibition upon PIP(2) depletion. Unlike the protein product of the major ROMK allele, these two variants are sensitive to the inhibitory affects of a G protein-coupled receptor, which stimulates PIP(2) hydrolysis. In summary, we have found that hypertension resistance sequence variants inhibit ROMK channel function by different mechanisms, providing new insights into the role of the channel in the maintenance of blood pressure.     

An ES-like pluripotent state in FGF-dependent murine iPS cells

Recent data demonstrates that stem cells can exist in two morphologically, molecularly and functionally distinct pluripotent states; a naïve LIF-dependent pluripotent state which is represented by murine embryonic stem cells (mESCs) and an FGF-dependent primed pluripotent state represented by murine and rat epiblast stem cells (EpiSCs). We find that derivation of induced pluripotent stem cells (iPSCs) under EpiSC culture conditions yields FGF-dependent iPSCs from hereon called FGF-iPSCs) which, unexpectedly, display naïve ES-like/ICM properties. FGF-iPSCs display X-chromosome activation, multi-lineage differentiation, teratoma competence and chimera contribution in vivo. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions.Characterization of the key molecular signalling pathways revealed FGF-iPSCs to depend on the Activin/Nodal and FGF pathways, while signalling through the JAK-STAT pathway is not required for FGF-iPS cell maintenance. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions.     

Structural properties and reactivity of N-terminal synthetic peptides of herpes simplex virus type 1 glycoprotein D by using antipeptide antibodies and group VII monoclonal antibodies.

To investigate the contribution of individual amino acids to the antigenicity of the N-terminal region of herpes simplex virus type 1 glycoprotein D, a series of 14 overlapping synthetic peptides within residues 1 to 30 were examined for their reactivity with monoclonal antibody LP14 (a group VII monoclonal antibody; in herpes simplex virus mutants resistant to LP14, arginine 16 is substituted by histidine) and two antipeptide antisera (antipeptide 9-21 and antipeptide 1-23). Maximal binding was achieved with peptides 9-21, 10-30, 9-30, and 8-30 and the chymotryptic fragment 9-17 of peptide 9-21, suggesting that a major antigenic site is located within residues 10 through 17. Lysine 10 was shown to be essential for high reactivity, either by binding directly to the antibody molecule or by stabilizing an ordered structure of the peptide. The importance of ordered structure was demonstrated by a decrease in reactivity after sodium dodecyl sulfate treatment of peptides 9-21 and 8-30.     

Effects of age and site quality on the distribution of biomass in Scots pine (Pinus sylvestris L.)

The distribution of the above-ground and below-ground biomass of Scots pine in southern Finland were investigated in trees of different ages (18–212 years) from two types of growth site. Secondly, some structural regularities were tested for their independence of age and growth site. Trees were sampled from dominant trees which could be expected to have a comparable position in stands of all ages. All stands were on sorted sediments. The biomass of the sample trees (18 trees) was divided into needles, branch sapwood and heartwood, stem sapwood and heartwood, stem bark, stump, large roots (diameter >20 cm), coarse roots (five classes) and fine roots. The amount of sapwood and heartwood was also estimated from the below-ground compartments. Trees on both types of growth site followed the same pattern of development of the relative shares of biomass compartments, although the growth rates were faster on the more fertile site. The relative amount of sapwood peaked after canopy closure, coinciding with the start of considerable heartwood accumulation. The relative amount of needles and fine roots decreased with age. The same was true of branches but to a lesser degree. The relative share of the below-ground section was independent of tree age. Foliage biomass and sapwood cross-sectional area were linearly correlated, but there were differences between the growth sites. Needle biomass was linearly correlated with crown surface area. The fine root to foliage biomass ratio showed an increasing trend with tree age.     

Photoperiodic induction of dormancy and freezing tolerance in Betula pubescens. Involvement of ABA and dehydrins

In many woody plants photoperiod signals the initiation of dormancy and cold acclimation. The photoperiod-specific physiological and molecular mechanisms have remained uncharacterised. The role of abscisic acid (ABA) and dehydrins in photope-riod-induced dormancy and freezing tolerance was investigated in birch, Betula pubescens Ehrh. The experiments were designed to investigate if development of dormancy and freezing tolerance under long-day (LD) and short-day (SD) conditions could be affected by manipulation of the endogenous ABA content, and if accumulation of dehydrin-like proteins was correlated with SD and/or the water content of the buds. Experimentally, the internal ABA content was increased by ABA application and by water stress treatment under LD, and decreased by blocking the synthesis of ABA with fluridone under SD. Additionally, high humidity (95% RH) was applied to establish if accidental water stress was involved in SD. ABA content was monitored by gas chromatography-mass spectrometry with selective ion monitoring (SIM). Short days induced a transient increase in ABA content, which was absent in 95% RH, whereas fluridone treatment decreased ABA. Short days induced a typical pattern of bud desiccation and growth cessation regardless of the treatment, and improved freezing tolerance except in the fluridone treatment. ABA content of the buds was significantly increased after spraying ABA on leaves and after water stress, treatments that did not induce cessation of growth and dormancy, but improved freezing tolerance. In addition to several constitutively produced dehydrins, two SD-specific proteins of molecular masses 34 and 36 kDa were found. Photoperiod- and experimentally-induced alterations in ABA contents affected freezing tolerance but not cessation of growth and dormancy. Therefore, involvement of ABA in the photoperiodic control of cold acclimation is more direct than in growth cessation and dormancy. As the typical desiccation pattern of the buds was found in all SD plants, and was not directly related to ABA content or to freezing tolerance, this pattern characterises the onset of photo-period-induced growth cessation and dormancy. The results provide evidence for the existence of various constitutively and two photoperiod-induced dehydrins in buds of birch, and reveal characteristics of dormancy and freezing tolerance that may facilitate further investigations of photoperiodic control of growth in trees.     

Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes

Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the species Bacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and the Clostridium lituseburense groups. Another probe was designed for the genera Streptococcus and Lactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectale group. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 × 10¹⁰ cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectale group-specific probe detected a mean of 7.2 × 10¹⁰ cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, and Streptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 × 10⁷ to 7 × 10⁸ per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.     

Onset of freezing tolerance in birch (Betula pubescens Ehrh.) involves LEA proteins and osmoregulation and is impaired in an ABA-deficient genotype

In many woody plants a short photoperiod triggers the onset of cold acclimation, but the nature of this process has remained obscure. We aimed to establish which physiological and genetic factors have a role in short-day-induced acclimation by comparing two types of birch, Betula pubescens Ehrh. and B. pubescens f. hibernifolia Ulv., the latter being unable to increase its abscisic acid (ABA) levels. In the wild type, short-day or natural autumn conditions in the field appeared to elevate the ABA levels before acclimation, which was accompanied by tissue desiccation, osmotic adjustments and accumulation of Group 2 LEA proteins [responsive to ABA (RAB) 16-like; 24, 30 and 33 kDa] and Group 4 LEA proteins [late embryogenesis abundant (LEA) 14-like; 19 kDa]. Under similar conditions the ABA-deficient birch showed reduced water loss, defective osmoregulation, absence of inducible Group 2 LEA proteins, and delayed or reduced tolerance to freezing. In contrast, both birch genotypes showed similar seasonal production patterns of Group 4 LEA proteins. Our results demonstrate that onset of cold acclimation in birch is based on multiple mechanisms, including molecular pathways that are typical of stress responses. ABA may be important for the accurate timing of cold acclimation in trees that are sensitive to photoperiod.