SLO-2 isoforms with unique Ca2+- and voltage-dependence characteristics confer sensitivity to hypoxia in C. elegans

Slo channels are large conductance K⁺ channels that display marked differences in their gating by intracellular ions. Among them, the Slo1 and C. elegans SLO-2 channels are gated by calcium (Ca²⁺), while mammalian Slo2 channels are activated by both sodium (Na⁺) and chloride (Cl⁻). Here, we report that SLO-2 channels, SLO-2a and a novel N-terminal variant isoform, SLO-2b, are activated by Ca²⁺ and voltage, but in contrast to previous reports they do not exhibit Cl⁻ sensitivity. Most importantly, SLO-2 provides a unique case in the Slo family for sensing Ca²⁺ with the high-affinity Ca²⁺ regulatory site in the RCK1 but not the RCK2 domain, formed through interactions with residues E319 and E487 (that correspond to D362 and E535 of Slo1, respectively). The SLO-2 RCK2 domain lacks the Ca²⁺ bowl structure and shows minimal Ca²⁺ dependence. In addition, in contrast to SLO-1, SLO-2 loss-of-function mutants confer resistance to hypoxia in C. elegans. Thus, the C. elegans SLO-2 channels possess unique biophysical and functional properties.     

Current progress in use of adipose derived stem cells in peripheral nerve regeneration

Unlike central nervous system neurons; those in the peripheral nervous system have the potential for full regeneration after injury. Following injury, recovery is controlled by schwann cells which replicate and modulate the subsequent immune response. The level of nerve recovery is strongly linked to the severity of the initial injury despite the significant advancements in imaging and surgical techniques. Multiple experimental model shave been used with varying successes to augment the natural regenerative processes which occur following nerve injury. Stem cell therapy in peripheral nerve injury may be an important future intervention to improve the best attainable clinical results. In particular adipose derived stem cells(ADSCs) are multipotent mesenchymal stem cells similar to bone marrow derived stem cells, which are thought to have neurotrophic properties and the ability to differentiate into multiple lineages. They are ubiquitous within adipose tissue; they can form many structures resembling the mature adult peripheral nervous system. Following early in vitro work; multiple small and large animal in vivo models have been used in conjunction with conduits, autografts and allografts to successfully bridge the peripheral nerve gap. Some of the ADSC related neuroprotective and regenerative properties have been elucidated however much work remains before a model can be used successfully in human peripheral nerve injury(PNI). This review aims to provide a detailed overview of progress made in the use of ADSC in PNI, with discussion on the role of a tissue engineered approach for PNI repair.     

Nonlinear mixed-effects branch diameter and length models for natural Dahurian larch (<Emphasis Type="Italic">Larix gmelini</Emphasis>) forest in northeast China

Key message

We developed the generalized branch diameter and length models using the multi-level nonlinear mixed-effects techniques for the natural Dahurian larch ( Larix gmelini ) forest in northeast China.

Abstract

Dahurian larch (Larix gmelini) is the most commercially cultivated timber species in northeastern China due to its ecological prevalence and its superior wood attribute. However, its timber quality was largely driven by the crown architecture, i.e., the number, size and distribution of branches. The majority of branch-level models in the literature are focused on planted forests, which have substantially different crown architecture than that grown in natural mixed forests. Therefore, the goal of this investigation was to develop branch diameter and length models for Dahurian larch that are grown in natural mixed forests. A multi-level nonlinear mixed-effects model technique, including the fixed-effects, random-effects, variance functions and correlation structures, was employed to develop the branch growth models. The results suggested that the cumulative branch diameter and length were both increased with the increases of branch depth into the crown. Diameter at breast height (DBH) had significant positive influences on the branch size; however, tree height (HT) produced negative influences on the branch size, i.e., larger DBH and smaller HT could lead to larger branch size. Model fitting and validation results confirmed that we should avoid developing over-complex models from the perspective of application. As for the branch diameter and length models in our study, addressing the stand and tree level effects as random component were quite reliable and accurate for predicting the branch growth process of Dahurian larch in northeastern China.

     

A Novel Cholinergic Action of Alcohol and the Development of Tolerance to That Effect in Caenorhabditis elegans

The Prp43 DExD/H-box protein is required for progression of the biochemically distinct pre-messenger RNA and ribosomal RNA (rRNA) maturation pathways. In Saccharomyces cerevisiae, the Spp382/Ntr1, Sqs1/Pfa1, and Pxr1/Gno1 proteins are implicated as cofactors necessary for Prp43 helicase activation during spliceosome dissociation (Spp382) and rRNA processing (Sqs1 and Pxr1). While otherwise dissimilar in primary sequence, these Prp43-binding proteins each contain a short glycine-rich G-patch motif required for function and thought to act in protein or nucleic acid recognition. Here yeast two-hybrid, domain-swap, and site-directed mutagenesis approaches are used to investigate G-patch domain activity and portability. Our results reveal that the Spp382, Sqs1, and Pxr1 G-patches differ in Prp43 two-hybrid response and in the ability to reconstitute the Spp382 and Pxr1 RNA processing factors. G-patch protein reconstitution did not correlate with the apparent strength of the Prp43 two-hybrid response, suggesting that this domain has function beyond that of a Prp43 tether. Indeed, while critical for Pxr1 activity, the Pxr1 G-patch appears to contribute little to the yeast two-hybrid interaction. Conversely, deletion of the primary Prp43 binding site within Pxr1 (amino acids 102–149) does not impede rRNA processing but affects small nucleolar RNA (snoRNA) biogenesis, resulting in the accumulation of slightly extended forms of select snoRNAs, a phenotype unexpectedly shared by the prp43 loss-of-function mutant. These and related observations reveal differences in how the Spp382, Sqs1, and Pxr1 proteins interact with Prp43 and provide evidence linking G-patch identity with pathway-specific DExD/H-box helicase activity.     

Requirement for the polarisome and formin function in Ssk2p-mediated actin recovery from osmotic stress in Saccharomyces cerevisiae

Osmotic stress induces activation of an adaptive mitogen-activated protein kinase pathway in concert with disassembly of the actin cytoskeleton by a mechanism that is not understood. We have previously shown that the conserved actin-interacting MAP kinase kinase kinase Ssk2p/MEKK4, a member of the high-osmolarity glycerol (HOG) MAPK pathway of Saccharomyces cerevisiae, mediates recovery of the actin cytoskeleton following osmotic stress. In this study, we have employed in vitro kinase assays to show that Ssk2p kinase activity is activated for the actin recovery pathway via a noncanonical, Ssk1p-independent mechanism. Our work also shows that Ssk2p requires the polarisome proteins Bud6p and Pea2p to promote efficient, polarized actin reassembly but that this requirement can be bypassed by overexpression of Ssk2p. Formin (BNI1 or BNR1) and tropomyosin functions are also required for actin recovery but, unlike for Bud6p and Pea2p, these requirements cannot be bypassed by overexpression of Ssk2p. These results suggest that Ssk2p acts downstream of Bud6p and Pea2p and upstream of tropomyosin to drive actin recovery, possibly by upregulating the actin nucleation activity of the formins.     

New investigations on hematoxylin, hematein, and hematein-aluminium complexes. II. Hematein-aluminium complexes and hemalum staining.

The absorption spectra of hematein-aluminium solutions have been recorded at various concentrations and pH values; the solutions were prepared using analytically pure hematein and potassium alum as aluminium source. In aqueous solution, four different hematein-aluminium complexes could be distinguished by absorption spectroscopy. In weakly acidic media we observed the violet 1:1 and 1:2 complexes HmAl (VII) and HmAl2(3) (VIII), and in strongly acidic solution the red 1:1 complex HmAl2 (IX). Whereas, in weakly alkaline solution the blue 1:1 complex HmAl0 (X) was detected. By change of the pH value the complexes were mutual interconverted. The dye complexes were characterized by their absorption spectra and molar extinction coefficients. We have stained HeLa cells with the complex solutions under different experimental conditions. In all cases the nuclear staining was intense whereas the staining of the cytoplasm was weak. The microspectra of the stained nuclei were recorded and compared with the absorption spectra of the complexes in solution. Thus it was possible to identify the bound dye species. After staining in acidic media, the cells were red to red-violet depending on the reaction conditions. The three cationic dye species VII, VIII, and IX were bound in varying amounts. After blueing in weakly acidic media or in water, only the violet dye complex VII was detected whereas, after blueing in weakly alkaline media, only the blue complex X has been observed. Enzymatic digestion experiments have shown that the dye complexes in the nuclei were bound to DNA while those in the cytoplasm and nucleoli were bound to RNA. The binding between the dye complexes and the nucleic acids is discussed.     

Loss of RAB-3/A in Caenorhabditis elegans and the mouse affects behavioral response to ethanol

The mechanisms by which ethanol induces changes in behavior are not well understood. Here, we show that Caenorhabditis elegans loss-of-function mutations in the synaptic vesicle-associated RAB-3 protein and its guanosine triphosphate exchange factor AEX-3 confer resistance to the acute locomotor effects of ethanol. Similarly, mice lacking one or both copies of Rab3A are resistant to the ataxic and sedative effects of ethanol, and Rab3A haploinsufficiency increases voluntary ethanol consumption. These data suggest a conserved role of RAB-3-/RAB3A-regulated neurotransmitter release in ethanol-related behaviors.     

Commonly invasive serotypes of Streptococcus pneumoniae trigger a reduced innate immune response compared with serotypes rarely responsible for invasive infection

Although there are more than 90 serotypes of Streptococcus pneumoniae (or pneumococcus), it is not understood why a small number of serotypes account for most invasive infections. To investigate the human innate immune response triggered by different pneumococcal serotypes, monocyte-derived macrophages were exposed to a group of commonly and rarely invasive pneumococcal clinical isolates and tumor necrosis factor (TNF)-alpha production was measured. Commonly invasive pneumococcal serotypes triggered significantly less TNF-alpha production than serotypes rarely responsible for invasive infection (P<0.004). These data indicate that one factor influencing the invasive potential of a pneumococcal serotype is the magnitude of innate immune-mediated TNF-alpha production triggered by exposure to the organism and suggest that the integrated host response generated against commonly invasive pneumococcal serotypes may be less effective than the response directed against rarely invasive serotypes.     

Noninvasive auto-photoreduction used as a tool for studying structural changes in heme-copper oxidases by FTIR spectroscopy

We demonstrate an efficient Fourier transform infrared (FTIR) spectroscopic method, termed "auto-photoreduction," that uses anaerobic photo-induced internal electron transfer to monitor reaction-initiated changes of heme-copper oxidases. It can be applied without the use of either expensive electrochemical equipment, or caged compounds, which cause significant background signals. At high irradiation power, carbon monoxide is released from high-spin heme a of cytochrome c oxidase and heme o from cytochrome bo(3). Photochemistry is initiated at wavelengths <355 nm, and the photochemical action spectrum has a maximum of 290 nm for cytochrome bo(3), which is consistent with the possible intermediate involvement of tyrosinate or an activated state of tyrosine. We propose that the final electron donors are proton channel water molecules. In the pH range of 4-9, the noninvasive auto-photoreduction method yields highly reproducible FTIR redox difference spectra within a broad range, resolving a number of vibrational changes outside the amide I region (1600-1640 cm(-1)). Furthermore, it provides details of redox-induced changes in the spectral region between 1600 and 1100 cm(-1). The auto-photoreduction method should be universally applicable to heme proteins.