Organization of connectin/titin filaments in sarcomeres of differentiating chicken skeletal muscle cells

Very long, elastic connectin/titin molecules position the myosin filaments at the center of a sarcomere by linking them to the Z line. The behavior of the connectin filaments during sarcomere formation in differentiating chicken skeletal muscle cells was observed under a fluorescent microscope using the antibodies to the N terminal (located in the Z line), C terminal (M line), and C zone (myosin filament) regions of connectin and was compared to the incorporation of -actinin and myosin into forming sarcomeres. In early stages of differentiating muscle cells, the N terminal region of connectin was incorporated into a stress fiber-like structure (SFLS) together with -actinin to form dots, whereas the C terminal region was diffusely distributed in the cytoplasm. When both the C and N terminal regions formed striations in young myofibrils, the epitope to the C zone of A-band region, that is the center between the A-I junction and the M-line, initially was diffuse in appearance and later formed definite striations. It appears that it took some time for the N and C terminal regions of connectin to form a regular organization in a sarcomere. Thus the two ends of the connectin filaments were first fixed followed by the specific binding of the middle portion onto the myosin filament during sarcomere formation.     

Genetic variability of foetal bovine myoblasts in primary culture

Selected strains of adult bovines and those which either have high muscle growth capacity or are double-muscled present particular characteristics of muscle fibres and collagen at slaughter that favour meat tenderness. For double-muscled bovines, it has been shown that these characteristics originated during foetal life. However, no studies have been done to determine the origin of muscle growth superiority in bovine with high muscle growth capacity compared to those with a low muscle growth capacity. Therefore, the objective of this study was to examine the proliferation and differentiation phases of myoblasts in primary culture taken from high and low muscle growth capacity foetuses at 110 days post-conception. These cultures were analysed on 1, 2, 3, 4, 6, 8, 10 days of culture. The proliferation phase was monitored by appropriate marker antibodies. The differentiation was studied by immunocytochemistry with specific antibodies for foetal, I, II (IIa and IIb), I and IIb, I and IIa myosin heavy chains (MHCs) and connectin respectively, and by immunoblotting with desmin antibody. A higher proliferation, a lower fusion and a delayed differentiation of the late markers namely MHCs fast (IIa and IIb) and connectin were shown in high muscle growth capacity foetuses compared to low capacity ones. The results indicate that the muscle growth superiority of high muscle growth capacity bovines seems, therefore, to have a similar foetal origin to that of double-muscled ones.     

An historical perspective of the discovery of titin filaments –Part 2

In 2017, a Special Issue of Biophysical Reviews was devoted to “Titin and Its Binding Partners. The issue contained a review: “An historical perspective of the discovery of titin filaments” by dos Remedios and Gilmour that was intended to be a history of the discovery of the giant protein titin, previously named connectin. The review took readers back to the earliest discovery of the so-called third filament component of skeletal and cardiac muscle sarcomeres and ended in 1969. Recently, my colleague Shin’ichi Ishiwata gently reminded me of two papers published in 1990 and 1993 that were unwittingly omitted from the original historical perspective. In the first paper (J Cell Biol 110:53–62, 1990), Funatsu et al. examined the elastic filaments in skeletal muscle using a combination of light and electron microscopy, but they also measured resting as well as passive stiffness mechanical measurements to establish that connectin (titin) is responsible for both stiffness and fiber tension. In the second paper (J Cell Biol 120:711–724, 1993), Funatsu et al. used permeabilised cardiac muscle myocytes (from rabbit papillary muscles) and focussed on filament ultrastructure using either freeze-substitution or deep-etched replica methods to visualise connectin/titin filaments in fibers with and without actin and myosin filaments.     

1H and 15N NMR resonance assignments and secondary structure of titin type I domains

Titin/connectin is a giant muscle protein with a highly modular architecture consisting ofmultiple repeats of two sequence motifs, named type I and type II. Type I modules have beensuggested to be intracellular members of the fibronectin type III (Fn3) domain family. Alongthe titin sequence they are exclusively present in the region of the molecule located in thesarcomere A-band. This region has been shown to interact with myosin and C-protein. Oneof the most noticeable features of type I modules is that they are particularly rich insemiconserved prolines, since these residues account for about 8% of their sequence. We havedetermined the secondary structure of a representative type I domain (A71) by 15N and 1HNMR. We show that the type I domains of titin have the Fn3 fold as proposed, consisting ofa three- and a four-stranded -sheet. When the two sheets are placed on top of each other toform the -sandwich characteristic of the Fn3 fold, 8 out of 10 prolines are found on the sameside of the molecule and form an exposed hydrophobic patch. This suggests that thesemiconserved prolines might be relevant for the function of type I modules, providing asurface for binding to other A-band proteins. The secondary structure of A71 was structurallyaligned to other extracellular Fn3 modules of known 3D structure. The alignment shows thattitin type I modules have closest similarity to the first Fn3 domain of Drosophila neuroglian.     

Cross-linking of connectin, an elastic protein in muscle

Following reduction with NaB³H₄, connectin, an elastic protein prepared from chicken muscle, was found to contain the reducible cross-links derived from lysine and hydroxylysine aldehydes. The aldimine form of lysinonorleucine is the most abundant reducible cross-link in this elastic protein. A smaller proportion of the reduced cross-link, histidino-hydroxymerodesmosine, is also detected. Since collagen contamination in the connectin preparation was if any negligible, it is concluded that connectin and connective tissue proteins, collagen and elastin, share common features of coss-linking.     

Amphioxus connectin exhibits merged structure as invertebrate connectin in I-band region and vertebrate connectin in A-band region

Connectin is an elastic protein found in vertebrate striated muscle and in some invertebrates as connectin-like proteins. In this study, we determined the structure of the amphioxus connectin gene and analyzed its sequence based on its genomic information. Amphioxus is not a vertebrate but, phylogenetically, the lowest chordate. Analysis of gene structure revealed that the amphioxus gene is approximately 430 kb in length and consists of regions with exons of repeatedly aligned immunoglobulin (Ig) domains and regions with exons of fibronectin type 3 and Ig domain repeats. With regard to this sequence, although the region corresponding to the I-band is homologous to that of invertebrate connectin-like proteins and has an Ig-PEVK region similar to that of the Neanthes sp. 4000K protein, the region corresponding to the A-band has a super-repeat structure of Ig and fibronectin type 3 domains and a kinase domain near the C-terminus, which is similar to the structure of vertebrate connectin. These findings revealed that amphioxus connectin has the domain structure of invertebrate connectin-like proteins at its N-terminus and that of vertebrate connectin at its C-terminus. Thus, amphioxus connectin has a novel structure among known connectin-like proteins. This finding suggests that the formation and maintenance of the sarcomeric structure of amphioxus striated muscle are similar to those of vertebrates; however, its elasticity is different from that of vertebrates, being more similar to that of invertebrates.     

Age-related changes in the reducible cross-links on connectin from human skeletal muscle.

After NaB³H₄-reduction of connectin from human skeletal muscle, the changes in the amounts of the reducible cross-links and specific radioactivity of this elastic protein were followed throughout the whole life-span from embryo to old age. The reducible cross-links, aldimine forms of lysinonorleucine and histidino-hydroxymerodesmosine, and unidentified reducible compounds, which were assumed to be cross-linking amino acids, were found to remarkably decrease with age. A progressive decrease in the incorporation of tritium into the reducible compounds was also observed. We conclude that the conversion of the reducible cross-links derived from lysine and hydroxylysine aldehydes to non-reducible compounds is an essential step in the maturation of connectin fibrils, similar to collagen fibrils.     

Connectin filaments link thick filaments and Z lines in frog skeletal muscle as revealed by immunoelectron microscopy

In an earlier study connectin, an elastic protein of striated muscle, was found to be associated with "gap filaments" originating from the thick filaments in the myofibril, but it was not clear whether it extends to Z lines or not (Maruyama, K., H. Sawada, S. Kimura, K. Ohashi, H. Higuchi, and Y. Umazume, 1984, J. Cell Biol., 99:1391-1397). In the present immunoelectron microscopic study using polyclonal antibodies against native connectin, we have concluded that the connectin structures are directly linked to Z lines from the thick (myosin) filaments in myofibrils of skinned fibers of frog skeletal muscle. There were five distinct antibody-binding stripes in each half of the A band and two stripes in the A-I junction region. Deposits of antibodies were recognized in I bands and Z lines. We suggest that connectin filaments run alongside the thick filaments, starting from a region approximately 0.15 micron from the center of the A band.     

Isolation and Structure of Protodestruxin from Metarrhizium anisopliae

µ-Calpain quickly split the α-connectin in myofibrils into β-connectin, and then produced a 1700-kDa component. Cathepsin D also split α-connectin into β-connectin, further degrading it to fragments smaller than the 1700-kDa component with increasing incubation time. The action of cathepsin D on the connectin molecule was distinctly different from that of, µ-calpain in terms of the splitting rate and manner. When freshly excised muscle was exposed to a temperature of 37°C, complete disappearance of connectin (α, β and 1700-kDa component) was observed within 36h. In contrast, at 2°C, about 75% of connectin was retained as β-form even after 3 weeks. The present data suggest that the degradation of connectin in muscle might be caused by, µ-calpain in the early stage of aging, and then with time, this action is replaced by m-calpain or cathepsin D. However, the possibility of other intrinsic proteases participating in the degradation of connectin still remains.     

Analysis of the reducible components of the muscle protein, connectin: absence of lysine-derived cross-links

After exhaustive salt extractions of rabbit and human skeletal muscle, the amino acid compositions of the residual proteins were similar to those reported for connectin. Complete removal of collagen contamination was achieved only after treatment of the connectin preparations with bacterial collagenase. On reduction with KB³H₄, the small amounts of lysine-derived reducible cross-links that were present in the initial connectin preparations were completely absent after treatment with collagenase. In adult human connectin some hexitol-lysine derivatives were present after reduction. These results indicate that, in contrast to previous reports, connectin does not participate in the same lysyl oxidase-mediated cross-linking system that occurs in collagen and elastin.     

Elastic filaments in situ in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments

To clarify the full picture of the connectin (titin) filament network in situ, we selectively removed actin and myosin filaments from cardiac muscle fibers by gelsolin and potassium acetate treatment, respectively, and observed the residual elastic filament network by deep-etch replica electron microscopy. In the A bands, elastic filaments of uniform diameter (6-7 nm) projecting from the M line ran parallel, and extended into the I bands. At the junction line in the I bands, which may correspond to the N2 line in skeletal muscle, individual elastic filaments branched into two or more thinner strands, which repeatedly joined and branched to reach the Z line. Considering that cardiac muscle lacks nebulin, it is very likely that these elastic filaments were composed predominantly of connectin molecules; indeed, anti-connectin monoclonal antibody specifically stained these elastic filaments. Further, striations of approximately 4 nm, characteristic of isolated connectin molecules, were also observed in the elastic filaments. Taking recent analyses of the structure of isolated connectin molecules into consideration, we concluded that individual connectin molecules stretched between the M and Z lines and that each elastic filament consisted of laterally-associated connectin molecules. Close comparison of these images with the replica images of intact and S1-decorated sarcomeres led us to conclude that, in intact sarcomeres, the elastic filaments were laterally associated with myosin and actin filaments in the A and I bands, respectively. Interestingly, it was shown that the elastic property of connectin filaments was not restricted by their lateral association with actin filaments in intact sarcomeres. Finally, we have proposed a new structural model of the cardiac muscle sarcomere that includes connectin filaments.     

The role of migration in the genetic structure of populations in temporally and spatially varying environments II. Island Models

The Island Model introduced by Sewall Wright (1951) has proven to be a useful construction for studying the interaction of genetic drift, population subdivision, and mutation. Interest in the model has recently increased because of its relevance to certain questions involving the rate of differentiation of sub-populations under the neutral allele hypothesis (e.g., Smith, 1970; Latter, 1973). It is perhaps the only realistic population structure in which the test for neutrality proposed by Lewontin and Krakauer (1973) is valid (Lewontin and Krakauer, 1975). If data from natural populations is to be compared to the predictions of the Island Model, it is desirable to have an alternative model with the same migration pattern but with natural selection operating. In this paper one such model will be introduced where the stochastic element comes from random fluctuations in the environment rather than from genetic drift. The model is a direct extension of the one in the previous paper in this series (Gillespie, 1975) which dealt with a population which is subdivided into two patches with restricted migration between them.     

The alternative splicing of <Emphasis Type="Italic">EAM8</Emphasis> contributes to early flowering and short-season adaptation in a landrace barley from the Qinghai-Tibetan Plateau

Key message

The early flowering of Lalu was determined to be due to a novel spontaneous eam8 mutation, which resulted in intron retention and the formation of a putative truncated protein.

Abstract

Barley is a staple crop grown over an extensive area in the Qinghai-Tibetan Plateau. Understanding the genetic mechanism for its success in a high altitude is important for crop improvement in marginal environments. Early flowering is a critical adaptive trait that strongly influences reproductive fitness in a short growing season. Loss-of-function mutations at the circadian clock gene EARLY MATURITY 8 (EAM8) promote rapid flowering. In this study, we identified a novel, spontaneous recessive eam8 mutant with an early flowering phenotype in a Tibetan barley landrace Lalu, which is natively grown at a high altitude of approximately 4000 m asl. The co-segregation analysis in a F₂ population derived from the cross Lalu (early flowering)?×?Diqing 1 (late flowering) confirmed that early flowering of Lalu was determined to be due to an allele at EAM8. The eam8 allele from Lalu carries an A/G alternative splicing mutation at position 3257 in intron 3, designated eam8.l; this alternative splicing event leads to intron retention and a putative truncated protein. Of the 134 sequenced barley accessions, which are primarily native to the Qinghai-Tibet Plateau, three accessions carried this mutation. The eam8.l mutation was likely to have originated in wild barley due to the presence of the Lalu haplotype in H. spontaneum from Tibet. Overall, alternative splicing has contributed to the evolution of the barley circadian clock and in the short-season adaptation of local barley germplasm. The study has also identified a novel donor of early-flowering barley which will be useful for barley improvement.

     

The tyramine binding site in the central nervous system: An overview

The [³H]Tyramine (TY) binding site is proposed as a high affinity marker of the membrane carrier for dopamine (DA) in synaptic vesicles from DA-rich brain regions. Under precise assay conditions, there is neither a consistent association of TY with the neuronal, cocaine-sensitive DA transporter, nor with mitochondrial or microsomal targets. TY-labeled sites have a high affinity for selected toxins such as the Parkinsonian agent MPP⁺ (1-methyl-4-phenylpyridinium ion), or drugs such as diphenylalkylamine Ca²⁺-channel antagonists. The MPP⁺/TY site interaction, which in the striatum leads to depletion of vesicular DA, occurs in dopaminergic as well as in noradrenergic regions, though with different kinetic profiles. TY-labeled carriers for DA and noradrenaline (NA) in respective vesicles seem to be different entities, which might result in a region-specific rate of toxin sequestration and/or release from heterogeneous vesicles. Whereas MPP⁺ is a potent competitive-type inhibitor of [³H]TY binding, prenylamine-like Ca²⁺-channel antagonists can compete with TY for the vesicle site, in a tetrabenazine- or reserpine-like manner, and also inhibit TY binding thanks to the extra-channel directed impairment of membrane bioenergetics they are proposed to provoke. This follows from the generally-accepted assumption that similar mechanisms are operational for secretory organelles in adrenals and CNS, and from the marked sensitivity of TY binding to miscellaneous energy-disrupting agents. A model is therefore proposed, depicting the TY-, DA- or MPP⁺-labeled, vesicle carrier, as a dimeric protein which may switch from the cytoplasm-oriented, recognition state, to the vesicle-oriented, transport state, thanks to the establishment of an H⁺-ATPase-supported, membrane protein electrochemical gradient.     

Lack of Phosphatidylglycerol Inhibits Chlorophyll Biosynthesis at Multiple Sites and Limits Chlorophyllide Reutilization in Synechocystis sp. Strain PCC 6803

The negatively charged lipid phosphatidylglycerol (PG) constitutes up to 10% of total lipids in photosynthetic membranes, and its deprivation in cyanobacteria is accompanied by chlorophyll (Chl) depletion. Indeed, radioactive labeling of the PG-depleted ΔpgsA mutant of Synechocystis sp. strain PCC 6803, which is not able to synthesize PG, proved the inhibition of Chl biosynthesis caused by restriction on the formation of 5-aminolevulinic acid and protochlorophyllide. Although the mutant accumulated chlorophyllide, the last Chl precursor, we showed that it originated from dephytylation of existing Chl and not from the block in the Chl biosynthesis. The lack of de novo-produced Chl under PG depletion was accompanied by a significantly weakened biosynthesis of both monomeric and trimeric photosystem I (PSI) complexes, although the decrease in cellular content was manifested only for the trimeric form. However, our analysis of ΔpgsA mutant, which lacked trimeric PSI because of the absence of the PsaL subunit, suggested that the virtual stability of monomeric PSI is a result of disintegration of PSI trimers. Interestingly, the loss of trimeric PSI was accompanied by accumulation of monomeric PSI associated with the newly synthesized CP43 subunit of photosystem II. We conclude that the absence of PG results in the inhibition of Chl biosynthetic pathway, which impairs synthesis of PSI, despite the accumulation of chlorophyllide released from the degraded Chl proteins. Based on the knowledge about the role of PG in prokaryotes, we hypothesize that the synthesis of Chl and PSI complexes are colocated in a membrane microdomain requiring PG for integrity.Photosynthetic membrane of oxygenic phototrophs has a unique lipid composition that has been conserved during billions of years of evolution from cyanobacteria and algae to modern higher plants. With no known exception, this membrane system always contains the uncharged glycolipids monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG) as well as the negatively charged lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG; Murata and Siegenthaler, 1998). Interestingly, it seems that PG is the only lipid completely essential for the oxygenic photosynthesis. The loss of DGDG has only a mild impact on the cyanobacterial cell (Awai et al., 2007), and as shown recently in the cyanobacterium Synechocystis sp. strain PCC 6803, both galactolipids can be in fact replaced by glucolipids (Awai et al., 2014). SQDG and PG are only minor lipid components, each accounting for 5% to 12% of total lipids (Murata and Siegenthaler, 1998). SQDG is dispensable, although its lack results in various defects (Yu et al., 2002; Aoki et al., 2004), but PG plays an essential role in both cyanobacterial cells and plant chloroplasts (Hagio et al., 2000; Babiychuk et al., 2003).The critical role of PG has been mostly connected to the function of PSII. In both cyanobacteria and plants, lack of PG impairs the stability of PSII complexes and the electron transport between primary and secondary quinone acceptors inside the PSII reaction center. As shown in Synechocystis sp., PG molecules stabilize PSII dimers and facilitate the binding of inner antenna protein CP43 within the PSII core (Laczkó-Dobos et al., 2008). Indeed, according to the PSII crystal structure, two PG molecules are located at the interface between CP43 and the D1-D2 heterodimer (Guskov et al., 2009). As a consequence, the PG depletion inhibits and destabilizes PSII complexes and also, impairs assembly of new PSII complexes, although all PSII subunits are still synthesized in the cell (Laczkó-Dobos et al., 2008).Despite the fact that the vital link between PG and PSII is now well established, the phenotypic traits of PG-depleted cells signal that there are other sites in the photosynthetic membrane requiring strictly PG molecules. In Synechocystis sp., lack of PG triggers rapid loss of trimeric PSI complexes (Domonkos et al., 2004; Sato et al., 2004), and because PSI complexes bind more than 80% of chlorophyll (Chl) in the Synechocystis sp. cell, the PG depletion is accompanied by a characteristic Chl bleaching (Domonkos et al., 2004). However, the reasons for this symptom are still unclear. Chl metabolism is tightly coordinated with synthesis, assembly, and degradation of photosystem complexes (for review, see Komenda et al., 2012b; Sobotka, 2014), and we have shown recently that the PSI complexes are the main sink for de novo Chl produced in cyanobacteria (Kopečná et al., 2012). Given the drastic decrease in PSI content in the PG-depleted cells, Chl biosynthesis must be directly or indirectly affected after the PG concentration in membranes drops below a critical value. Although it was recently suggested that galactolipid and Chl biosyntheses are coregulated during chloroplast biogenesis (Kobayashi et al., 2014), a response of the Chl biosynthetic pathway to the altered lipid content has not been examined.To investigate Chl metabolism during PG starvation, we used the Synechocystis sp. ΔpgsA mutant, which is unable to synthesize PG (Hagio et al., 2000). The advantage of using the ΔpgsA strain is in its ability to utilize exogenous PG from growth medium, which allows monitoring of phenotypic changes from a wild type-like situation to completely PG-depleted cells. Chl biosynthesis shares the same metabolic pathway with heme and other tetrapyrroles. At the beginning of tetrapyrrole biosynthesis, the initial precursor, 5-aminolevulinic acid (ALA), is made from Glu through glutamyl-tRNA and subsequently converted in several steps to protoporphyrin IX. The pathway branches at the point where protoporphyrin IX is chelated by magnesium to produce Mg-protoporphyrin IX, the first intermediate on the Chl branch. This step is catalyzed by Mg-chelatase, a multisubunit enzyme that associates relatively weakly with the membrane; however, all following enzymes downward in the pathway are almost exclusively bound to membranes (Masuda and Fujita, 2008; Kopečná et al., 2012). The last enzyme of the Chl pathway, Chl synthase, is an integral membrane protein that attaches a phytyl chain to the last intermediate chlorophyllide (Chlide) to finalize Chl formation (Oster et al., 1997; Addlesee et al., 2000). According to current views, Chl synthase should also be involved in reutilization of Chl molecules from degraded Chl-binding proteins, which includes dephytylation and phytylation of Chl molecules with Chlide as an intermediate (Vavilin and Vermaas, 2007).In this study, we show a complex impact of PG deficiency on Chl metabolism. The lack of PG inhibited Chl biosynthesis at the two different steps: first, it drastically reduced formation of the initial precursor ALA, and second, it impaired the Mg-protoporphyrin methyl ester IX (MgPME) cyclase enzyme catalyzing synthesis of protochlorophyllide (Pchlide). The diminished rate of Chl formation was accompanied by impaired synthesis of both trimeric and monomeric PSI complexes and accumulation of a PSI monomer associated with the CP43 subunit of PSII. We also showed that the PG-depleted cells accumulated Chlide, originating from dephytylation of existing Chl, which suggests an inability to reutilize Chl for the PSI synthesis. We discuss a scenario that the Chl biosynthesis and synthesis of core PSI subunits are colocated in PG-enriched membrane microdomains.     

Roles of SCaBP8 in salt stress response

The tissue-preferential distributed calcium sensors, SOS3 and SCaBP8, play important roles in SOS pathway to cope with saline conditions. Both SOS3 and SCaBP8 interact with and activate SOS2. However the regulatory mechanism for SOS2 activation and membrane recruitment by SCaBP8 differs from SOS3. SCaBP8 is phosphorylated by SOS2 at plasma membrane (PM) under salt stress. This phosphorylation anchors the SCaBP8-SOS2 complex on plasma membrane and activates PM Na⁺/H⁺ anti-porter, such as SOS1. Here, we describe that SOS2 has high binding affinity and catalytic efficiency to SCaBP8, suggesting that phosphorylation of SCaBP8 by SOS2 perhaps occurs rapidly in salt condition. SCaBP8 is also phosphorylated by PKS5 (SOS2-like Protein Kinase5) which negatively regulates PM H⁺-ATPase activity and functions in plant alkaline tolerance, providing a clue to roles of SCaBP8 in both salt and alkaline tolerance. SOS2 interacts with SOS3 and SCaBP8 with its FISL motif at C-terminus. However, luciferase activity complement assay indicates that SOS2 N-terminal is also essential for interacting with these proteins in plant.Key words: calcium signal, kinase activity, luciferase complementDue to their sessile nature, plants have developed elaborate strategies to deal with a number of environmental challenges. One overwhelming constraint is high salinity in the soil, which inhibits plant growth and decreases the agricultural productivity. Efflux and/or sequestering of sodium ion to apoplastic space/vacuolar are well-known cellular mechanisms that plants protect them from saline stress.¹ Recently identified SOS (salt overly sensitive) pathway plays critical roles in maintaining ion homeostasis in response to high salinity.² Two calcium sensors, SOS3 and SCaBP8 (SOS3-like calcium binding protein8), perceive cytosolic calcium signature triggered by salt, interact with and activate a Thr/Ser protein kinase, SOS2 and recruit it to the plasma membrane. Then, the formed SOS3-SOS2 or SCaBP8-SOS2 complex activates a PM Na⁺/H⁺ anti-porter, SOS1.^2–4 Moreover, SOS2 also regulates vascular Na⁺/H⁺ antiporter activity.⁵ Previously, we reported that SCaBP8 and SOS3 function distinctly in activation of SOS2.³ For instance, N-terminal myristoylation of SOS3 plays an important role in salt tolerance.⁶ However, there is no consensus myristoylated motif in SCaBP8. Instead, an N-terminal hydrophobic domain is sufficient to facilitate the association of SCaBP8 to plasma membrane.³ In addition, SCaBP8 is phosphorylated by SOS2 under salt stress and this phosphorylation stabilizes the interaction of SOS2 and SCaBP8.⁴ In this report, we describe that SCaBP8 possibly is rapidly phosphorylated by SOS2 under salt stress and also phosphorylated by another stress responsible protein kinase, implying additional roles of SCaBP8 in stress responses.     

Widespread Head-to-Head Hydrocarbon Biosynthesis in Bacteria and Role of OleA

Previous studies identified the oleABCD genes involved in head-to-head olefinic hydrocarbon biosynthesis. The present study more fully defined the OleABCD protein families within the thiolase, α/β-hydrolase, AMP-dependent ligase/synthase, and short-chain dehydrogenase superfamilies, respectively. Only 0.1 to 1% of each superfamily represents likely Ole proteins. Sequence analysis based on structural alignments and gene context was used to identify highly likely ole genes. Selected microorganisms from the phyla Verucomicrobia, Planctomyces, Chloroflexi, Proteobacteria, and Actinobacteria were tested experimentally and shown to produce long-chain olefinic hydrocarbons. However, different species from the same genera sometimes lack the ole genes and fail to produce olefinic hydrocarbons. Overall, only 1.9% of 3,558 genomes analyzed showed clear evidence for containing ole genes. The type of olefins produced by different bacteria differed greatly with respect to the number of carbon-carbon double bonds. The greatest number of organisms surveyed biosynthesized a single long-chain olefin, 3,6,9,12,15,19,22,25,28-hentriacontanonaene, that contains nine double bonds. Xanthomonas campestris produced the greatest number of distinct olefin products, 15 compounds ranging in length from C₂₈ to C₃₁ and containing one to three double bonds. The type of long-chain product formed was shown to be dependent on the oleA gene in experiments with Shewanella oneidensis MR-1 ole gene deletion mutants containing native or heterologous oleA genes expressed in trans. A strain deleted in oleABCD and containing oleA in trans produced only ketones. Based on these observations, it was proposed that OleA catalyzes a nondecarboxylative thiolytic condensation of fatty acyl chains to generate a β-ketoacyl intermediate that can decarboxylate spontaneously to generate ketones.There is currently great interest in elucidating the means by which microbes produce nongaseous hydrocarbons for use as specialty chemicals and fuels (8, 40). While many details remain to be revealed, there appear to be several different pathways by which microbes biosynthesize long-chain hydrocarbons. The most studied of the pathways (16) involves the condensation of isoprene units to generate hydrocarbons with a multiple of five carbon atoms (C₁₀, C₁₅, C₂₀, etc.). A more obscure biosynthetic route is a reported decarbonylation of fatty aldehydes to generate a C_n−1 hydrocarbon chain (19). A third mechanism that has received some attention is what has been denoted head-to-head condensation of fatty acids (2, 3, 4, 8, 64). In this pathway, the hydrocarbons are described to arise from the formation of a carbon-to-carbon bond between the carboxyl carbon of one fatty acid and the α-carbon of another fatty acid (3). This condensation results in a particular type of hydrocarbon with chain lengths of C₂₃ to C₃₃ and containing one or more double bonds. One double bond involves the median carbon in the chain at the point of fatty acid condensation. An example of this overall biosynthetic pathway leading to the formation of specific C₂₉ olefinic hydrocarbon isomers from fatty acid precursors has been demonstrated in vivo (61, 63, 64) and in vitro (4, 8).The condensation, elimination of carbon dioxide, and loss of the other carboxyl group oxygen atoms likely require multiple enzyme-catalyzed reactions. Recent patent applications by L. Friedman et al. describe a role for three or four proteins in this biosynthetic pathway (18 September 2008, WO2008/113041; 4 December 2008, WO2008/147781). Most recently, Beller et al. demonstrated the requirement for three genes from Micrococcus luteus in the biosynthesis of long-chain olefins (8). That study also demonstrated in vitro production of olefins by recombinant proteins in the presence of crude cell extracts from Escherichia coli. In another study, Shewanella oneidensis strain MR-1 was shown to produce a head-to-head hydrocarbon (59). A cluster of four genes, oleABCD, was shown to be involved in olefin biosynthesis by that organism.While genetic and biochemical data have provided evidence for Ole proteins producing long-chain olefins in M. luteus and S. oneidensis, there are many outstanding details of the biosynthesis that remain to be elucidated. Moreover, the extent to which microbial and other species produce head-to-head olefins is unclear. A recent patent application by Friedman and Rude (WO2008/113041) presented tables listing genes homologous to the ole genes described by Beller et al. (8). However, the homologs identified included genes from mouse and tree frog, organisms not known to produce head-to-head hydrocarbons. Additionally, hydrocarbon biosynthetic genes from Arthrobacter sp. FB24 were claimed, and that strain was later shown not to produce hydrocarbons under identical conditions for which other Arthrobacter strains did (22). In that context, the present study closely examined the protein sequence families of Ole proteins and the configurations of putative ole genes within genomes to identify those most likely to be involved in head-to-head hydrocarbon biosynthesis. This was followed by experimental testing for the presence of long-chain head-to-head hydrocarbons in representative bacteria from diverse phyla. This study also found that, of closely related bacteria, some produce head-to-head hydrocarbons and others do not.A previous publication investigated in vitro olefin biosynthesis from myristyl-coenzyme A (CoA) (8). That study showed ketone and olefin biosynthesis in vitro and proposed a mechanism requiring the participation of ancillary proteins not encoded in the oleABCD gene cluster. The mechanism proposed fatty acyl oxidation to generate a β-keto acid that is the substrate for the OleA protein.In fact, different mechanisms have been suggested previously for the biosynthesis of head-to-head olefins (3; Friedman and Rude, WO2008/113041; Friedman and Da Costa, WO2008/147781), and different roles for the OleA protein have been proposed (8; Friedman and Rude, WO2008/113041; Friedman and Da Costa, WO2008/147781). It is not possible to deduce the olefinic biosynthetic pathway or individual reaction types based on protein sequence alignments alone, because this pathway is unique, differing markedly from isoprenoid or decarbonylation hydrocarbon biosynthesis pathways. Moreover, the individual Ole proteins are each homologous to proteins that collectively catalyze diverse reactions. In that context, we (i) initiated a more detailed study of the Ole protein superfamilies, (ii) identified likely olefin (ole) biosynthetic genes out of thousands of homologs, (iii) experimentally tested bacteria from different phyla for long-chain olefins, (iv) developed insights into the role of OleA in head-to-head olefin biosynthesis, and (v) propose an alternative mechanism for head-to-head condensation of fatty acyl groups.     

The neural basis of a sensory filter in the Jamming Avoidance Response: No grandmother cells in sight

Summary The Jamming Avoidance Response (JAR), during which weakly electric fish modulate their electric organ discharge rate in response to a foreign electric signal of nearly the same frequency is strongest for frequency differences (f s) between 3–8 Hz. We have searched for neural correlates of this behavioral specificity. Single unit recordings in the anterior lateral line ganglion (ALLG), the posterior lateral line lobe (PLLL) and the torus semicircularis (TS) ofEigenmannia virescens were made during electrical stimulation simulating jamming by a nearby conspecific.Contrary to previously published reports (Scheich 1974, 1977) we conclude that f specificity does not lie in a single class of receptors or higher-order units in the PLLL tuned to the most effective f s. No tuning is seen at the receptor level of the PLLL. Specificity seems to be a population effect first visible at the level of the torus semicircularis, with individual units responding most strongly to different f s, but with most units tuned to approximately + and-4 Hz. By having cells tuned to a variety of f s but occurring in proportions corresponding to the observed behavior (and the degree to which f s impair electrolocation), animals would be better equipped to carry out other tasks such as detection of relative motion of objects in space and would also be better able to read complex stimuli corresponding to the more usual case of simultaneous jamming from several conspecifics (Partridge and Heiligenberg 1980).Units in the PLLL show slight differences in the timing of their firing to jamming signals presented at a frequency slightly above (+f) the fish's pacemaker frequency compared to those presented at a frequency slightly below (–f) (Scheich 1977). Firing pattern within the beat cycle produced by interaction of the fish's EOD, or an electrical mimic, S₁, and the foreign signal, S₂, is largely unaffected by the field orientation of the jamming signal. In the torus, by contrast, two classes of units are encountered which completely reverse the pattern of their firing within the beat cycle if the sign of the f is reversed. And, unlike the PLLL cells, those in TS respond differentially to different stimulus field geometries. Units of class 1 appear to compare T-unit input from different sites on the body surface (Heiligenberg and Bastian 1980) whereas those of class 2 additionally appear to receive input from E- and I-units in the PLLL. Abbreviations: see MethodsThis study was supported by grants from the National Science Foundation and the National Institutes of Health.     

Selection of microorganisms which produce raw-starch degrading enzymes

Summary Microorganisms which produce strong raw-starch degrading enzymes were isolated from soil using a medium containing a unique carbon source, -amylase resistant starch (-RS), which is insoluble in water and hardly digested with Bacillus amyloliquefaciens -amylase. Among the isolates, three strains showing high activities were characterized. Two of them, K-27 (fungus) and K-28 (yeast), produced -amylase and glucoamylase, and the final product from starch was only glucose. The third strain, K-2, was a bacterium and produced -amylase, which produced glucose and malto-oligosaccharides from starch. The enzyme preparation of these strains degraded raw corn starch rapidly.