Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets

Star-PAP is a nuclear non-canonical poly(A) polymerase (PAP) that shows specificity toward mRNA targets. Star-PAP activity is stimulated by lipid messenger phosphatidyl inositol 4,5 bisphoshate (PI4,5P₂) and is regulated by the associated Type I phosphatidylinositol-4-phosphate 5-kinase that synthesizes PI4,5P₂ as well as protein kinases. These associated kinases act as coactivators of Star-PAP that regulates its activity and specificity toward mRNAs, yet the mechanism of control of these interactions are not defined. We identified a phosphorylated residue (serine 6, S6) on Star-PAP in the zinc finger region, the domain required for PIPKIα interaction. We show that S6 is phosphorylated by CKIα within the nucleus which is required for Star-PAP nuclear retention and interaction with PIPKIα. Unlike the CKIα mediated phosphorylation at the catalytic domain, Star-PAP S6 phosphorylation is insensitive to oxidative stress suggesting a signal mediated regulation of CKIα activity. S6 phosphorylation together with coactivator PIPKIα controlled select subset of Star-PAP target messages by regulating Star-PAP-mRNA association. Our results establish a novel role for phosphorylation in determining Star-PAP target mRNA specificity and regulation of 3′-end processing.     

Simultaneous saccharification and fermentation of straw to ethanol using the thermotolerant yeast strain Kluyveromyces marxianus imb3

The thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 was grown at 45°C on media containing 2, 4 and 6 % (w/v) pulverized barley straw and supplemented with 2% (v/v) cellulase. Maximum ethanol concentrations produced were 2, 3 and 3.6g/l, respectively. When the pulverized straw was replaced by NaOH pretreated straw (at 2, 4 and 6% (w/v); based on original untreated straw), ethanol concentrations increased to maxima of 3.9, 8, and 12g/l, respectively. The ethanol yields amount to 20g ethanol from 100g of straw.     

A mutation in the pore of the sodium channel alters gating.

Ion permeation and channel gating are classically considered independent processes, but site-specific mutagenesis studies in K channels suggest that residues in or near the ion-selective pore of the channel can influence activation and inactivation. We describe a mutation in the pore of the skeletal muscle Na channel that alters gating. This mutation, I-W53C (residue 402 in the mu 1 sequence), decreases the sensitivity to block by tetrodotoxin and increases the sensitivity to block by externally applied Cd2+ relative to the wild-type channel, placing this residue within the pore near the external mouth. Based on contemporary models of the structure of the channel, this residue is remote from the regions of the channel known to be involved in gating, yet this mutation abbreviates the time to peak and accelerates the decay of the macroscopic Na current. At the single-channel level we observe a shortening of the latency to first opening and a reduction in the mean open time compared with the wild-type channel. The acceleration of macroscopic current kinetics in the mutant channels can be simulated by changing only the activation and deactivation rate constants while constraining the microscopic inactivation rate constants to the values used to fit the wild-type currents. We conclude that the tryptophan at position 53 in the domain IP-loop may act as a linchpin in the pore that limits the opening transition rate. This effect could reflect an interaction of I-W53 with the activation voltage sensors or a more global gating-induced change in pore structure.     

Evolutionary relatedness of some primate models of Plasmodium

Primate--and, specifically, monkey--malaria infections are commonly used for understanding the pathology of and immune response to the human disease because they are thought to resemble most closely the host-parasite relationship found in humans. Plasmodium cynomolgi is used extensively as a model for the human parasite, P. vivax, and P. knowlesi is used primarily as a model for the development of erythrocytic-stage vaccines. Both of these simian parasites can naturally infect man, resulting in mildly symptomatic episodes of the disease. The phylogenetic relationship between these two simian parasites and previously characterized Plasmodium species, including P. vivax, was examined by comparison of the asexually expressed small- subunit ribosomal RNA genes. Our analysis confirmed that P. vivax is most closely related to P. cynomolgi and that it remains an appropriate model of the human pathogen. Furthermore, with P. knowlesi and P. fragile, these two species form a group of closely related species, distant from other Plasmodium species. What is considered to be the most ancient of the human malaria pathogens, P. malariae, was also included in the analysis and does not group at all with other simian or human parasites.      

Evolution and phylogenetic information content of the ITS-1 region in the tiger beetle Cicindela dorsalis

Sequence divergence in the internal transcribed spacer region 1 (ITS-1) of the ribosomal DNA locus was assessed in subspecies of the coastal North American tiger beetle, Cicindela dorsalis. The spacer region was amplified using the polymerase chain reaction and cloned for sequencing. Of a total of 50 clones obtained from 12 specimens, 42 clones were different in at least one nucleotide position. In a parsimony analysis of these sequences, the main phylogenetic distinction was found to separate sequences from the Gulf of Mexico and the Atlantic Ocean. Within these two assemblages phylogenetic resolution was low, and the variation within individuals was almost as high as the variation within the entire lineage. The pattern of sequence variation suggests the existence of two forms of the ITS-1 that are maintained on different chromosomes. Polymorphisms of limited geographical distribution could be detected, and 41 additional clones were partly sequenced, to assess the geographic distribution of these polymorphisms in more detail. In a population aggregation analysis, the geographic pattern of ITS-1 distribution was basically congruent with that obtained in earlier studies from mitochondrial DNA in the same C. dorsalis populations.      

The effect of atrial natriuretic factor on force development in rat cardiac trabeculae

Studies in single cardiac muscle cells have demonstrated that atrial natriuretic factor decreases the L-type calcium current. Recent investigations in human atrial cells have also demonstrated that atrial natriuretic factor causes a voltage-dependent reduction in sodium channel activity and thus may reduce intracellular calcium via decreased activity of the sodium-calcium exchange mechanism. By reducing intracellular calcium, atrial natriuretic factor may have a negative inotropic effect on cardiac muscle. To characterize the effect of atrial natriuretic factor on the development of force, we studied the force-sarcomere length relationship in 11 right ventricular rat trabeculae, both before and after exposure of the muscles to increasing concentrations of atrial natriuretic factor. Sarcomere length was measured by laser diffraction techniques and controlled by a servomotor system. The addition of atrial natriuretic factor to the superfusion solution, at concentrations of 10(-9)-10(-7) M, increased stimulus threshold, reduced peak twitch force in a dose-dependent manner by 38% (maximum), and reduced time to peak twitch force by 15% (maximum). Incubation of muscle preparations with concentrations of atrial natriuretic factor below 10(-9) M had no effect on force generation. The negative inotropic effect of atrial natriuretic factor was associated with a change in the shape of the force-sarcomere length relationship, similar to a reduction of the extracellular calcium concentration. ANF (10(-7) M) had no effect on the rate of decay of force following post extra-systolic potentiation. These observations are consistent with the assumption that the negative inotropic effect of atrial natriuretic factor is mediated by reduction of calcium entry into the cardiac cell.     

A model of propagating calcium-induced calcium release mediated by calcium diffusion

The effect of sudden local fluctuations of the free sarcoplasmic [Ca++]i in cardiac cells on calcium release and calcium uptake by the sarcoplasmic reticulum (SR) was calculated with the aid of a simplified model of SR calcium handling. The model was used to evaluate whether propagation of calcium transients and the range of propagation velocities observed experimentally (0.05-15 mm s(-1)) could be predicted. Calcium fluctuations propagate by virtue of focal calcium release from the SR, diffusion through the cytosol (which is modulated by binding to troponin and calmodulin and sequestration by the SR), and subsequently induce calcium release from adjacent release sites of the SR. The minimal and maximal velocities derived from the simulation were 0.09 and 15 mm s(-1) respectively. The method of solution involved writing the diffusion equation as a difference equation in the spatial coordinates. Thus, coupled ordinary differential equations in time with banded coefficients were generated. The coupled equations were solved using Gear's sixth order predictor-corrector algorithm for stiff equations with reflective boundaries. The most important determinants of the velocity of propagation of the calcium waves were the diastolic [Ca++]i, the rate of rise of the release, and the amount of calcium released from the SR. The results are consistent with the assumptions that calcium loading causes an increase in intracellular calcium and calcium in the SR, and an increase in the amount and rate of calcium released. These two effects combine to increase the propagation velocity at higher levels of calcium loading.     

A new practical tool for deriving a functional signature for herbaceous vegetation

Hypothesis: For any one time and place a ‘functional signature’ can be derived for a sample of herbaceous vegetation in a way that concisely represents the balance between the different clusters of functional attributes that are present among component species. Methods: We developed a spreadsheet‐based tool for calculating functional signatures within the context of the C‐S‐R system of plant functional types. We used the tool to calculate and compare signatures for specimen British vegetation samples which differed in management regime and location in time. Conclusion: The integrative power of the ‘C‐S‐R signature’ is useful in comparative studies involving widely differing samples. Movements in the signature can be used to indicate degree of resistance, resilience, eutrophication and dereliction. Systems of plant functional types other than C‐S‐R might also be approached in this way. Availability: The tool can be downloaded free of charge from the first author's web pages or from the journal's electronic archive.     

2-hydroxyoleic acid affects cardiomyocyte [Ca2+]i transient and contractility in a region-dependent manner

Monounsaturated fatty acids such as oleic acid are cardioprotective, modify the physicochemical properties of cardiomyocyte membranes, and affect the electrical stability of these cells by regulating the conductance of ion channels. We have designed a nonhydrolysable oleic acid derivative, 2-hydroxyoleic acid (2-OHOA), which regulates membrane lipid structure and cell signaling, resulting in beneficial cardiovascular effects. We previously demonstrated that 2-OHOA induces PKA activation and PKCalpha translocation to the membrane; both pathways are thought to regulate transient outward K(+) current (I(to)) depending on the stimulus and the species used. This study was designed to investigate the effect of 2-OHOA on isolated cardiomyocytes. We examined the dose- and time-dependent effect of 2-OHOA on cytosolic Ca(2+) concentration ([Ca(2+)](i)) transient and contraction of myocytes isolated from different parts of the rat ventricular myocardium. Although this drug had no effect on [Ca(2+)](i) transient and cell shortening in myocytes isolated from the septum, it increased (up to 95%) [Ca(2+)](i) transient and cell shortening in subpopulations of myocytes from the right and left ventricles. The pattern of the effects of 2-OHOA was similar to that observed following the application of the I(to) blocker 4-aminopyridine, suggesting that the drug may act on this channel. Unlike the effect of 2-OHOA on [Ca(2+)](i) transient and cell shortening, PKCalpha translocation to membranes was not region specific. Thus 2-OHOA-induced effects on [Ca(2+)](i) transients and cell shortening are likely related to reductions in I(to) function, but PKCalpha translocation does not seem to play a role. The present results indicate that 2-OHOA selectively increases myocyte inotropic responsiveness, which could underlie its beneficial cardiovascular effects.