Contribution of biofilm‐dwelling consumers to pelagic–benthic coupling in a large river

1. Over the course of this 17‐month study, we assessed the potential loss of plankton (bacteria, algae, heterotrophic flagellates) to consumers (ciliates and rotifers) within mature biofilms established on natural substrata exposed to the main current of the River Rhine (Germany). Once a month, in flow cells in a bypass system to the River Rhine, we measured the clearance rates of the biofilm‐associated consumers on the different groups within the natural plankton. 2. Ciliates were the most dominant consumers, among which planktivorous groups, particularly peritrichs and (in spring and summer) heterotrichs dominated. Consumer biomass varied with season, with the highest density occurring directly after the appearance of the phytoplankton spring peak. 3. Clearance rates on plankton ranged from 96 to 565 L m^?2 d^?1 for bacteria and 66–749 L m^?2 d^?1 for algae, with a preference for algae in summer and for bacteria in winter. This pattern coincided with seasonal changes in the structures of the grazer communities. The consumers (both ciliates and rotifers with total standing stocks ranging between 19 and 572 mg C m^?2) imported a substantial amount of organic matter (between 15 and 137 mg C m^?2 d^?1) into the biofilm. 4. These results highlight the potential importance of consumers in the biofilm as a trophic link between the plankton and the benthos, a function that has hitherto mostly been attributed to filter‐feeding bivalves. In contrast to bivalves, the biofilm‐dwelling consumers show a more dynamic response towards the plankton density and composition. Such dynamic components need to be considered when estimating total plankton consumption by the benthos.     

Structure and Disorder in an Unfolded State under Nondenaturing Conditions from Ensemble Models Consistent with a Large Number of Experimental Restraints

Obtaining detailed structural models of disordered states of proteins under nondenaturing conditions is important for a better understanding of both functional intrinsically disordered proteins and unfolded states of folded proteins. Extensive experimental characterization of the drk N-terminal SH3 domain unfolded state has shown that, although it appears to be highly disordered, it possesses significant nonrandom secondary and tertiary structure. In our previous attempts to generate structural models of the unfolded state using the program ENSEMBLE, we were limited by insufficient experimental restraints and conformational sampling. In this study, we have vastly expanded our experimental restraint set to include ¹H-¹⁵N residual dipolar couplings, small-angle X-ray scattering measurements, nitroxide paramagnetic relaxation enhancements, O₂-induced ¹³C paramagnetic shifts, hydrogen-exchange protection factors, and ¹⁵N R₂ data, in addition to the previously used nuclear Overhauser effects, amino terminal Cu²⁺-Ni²⁺ binding paramagnetic relaxation enhancements, J-couplings, chemical shifts, hydrodynamic radius, and solvent accessibility restraints. We have also implemented a new ensemble calculation methodology that uses iterative conformational sampling and seeks to calculate the simplest possible ensemble models. As a result, we can now generate ensembles that are consistent with much larger experimental data sets than was previously possible. Although highly heterogeneous and having broad molecular size distributions, the calculated drk N-terminal SH3 domain unfolded-state ensembles have very different properties than expected for random or statistical coils and possess significant nonnative α-helical structure and both native-like and nonnative tertiary structure.     

Entropic elastic processes in protein mechanisms. II. Simple (passive) and coupled (active) development of elastic forces

The first part of this review on entropic elastic processes in protein mechanisms (Urry, 1988) demonstrated with the polypentapeptide of elastin (Val¹-Pro²-Gly³-Val⁴-Gly⁵)_n that elastic structure develops as the result of an inverse temperature transition and that entropic elasticity is due to internal chain dynamics in a regular nonrandom structure. This demonstration is contrary to the pervasive perspective of entropic protein elasticity of the past three decades wherein a network of random chains has been considered the necessary structural consequence of the occurrence of dominantly entropic elastomeric force. That this is not the case provides a new opportunity for understanding the occurrence and role of entropic elastic processes in protein mechanisms. Entropic elastic processes are considered in two classes: passive and active. The development of elastomeric force on deformation is class I (passive) and the development of elastomeric force as the result of a chemical process shifting the temperature of a transition is class II (active). Examples of class I are elastin, the elastic filament of muscle, elastic force changes in enzyme catalysis resulting from binding processes and resulting in the straining of a scissile bond, and in the turning on and off of channels due to changes in transmembrane potential. Demonstration of the consequences of elastomeric force developing as the result of an inverse temperature transition are seen in elastin, where elastic recoil is lost on oxidation, i.e., on decreasing the hydrophobicity of the chain and shifting the temperature for the development of elastomeric force to temperatures greater than physiological. This is relevant in general to loss of elasticity on aging and more specifically to the development of pulmonary emphysema. Since random chain networks are not the products of inverse temperature transitions and the temperature at which an inverse temperature transition occurs depends on the hydrophobicity of the polypeptide chain, it now becomes possible to consider chemical processes for turning elastomeric force on and off by reversibly changing the hydrophobicity of the polypeptide chain. This is herein called mechanochemical coupling of the first kind; this is the chemical modulation of the temperature for the transition from a less-ordered less elastic state to a more-ordered more elastic state. In the usual considerations to date, development of elastomeric force is the result of a standard transition from a more-ordered less elastic state to a less-ordered more elastic state. When this is chemically modulated, it is herein called mechanochemical coupling of the second kind. For elastin and the polypentapeptide of elastin, since entropic elastomeric force results on formation of a regular nonrandom structure and thermal randomization of chains results in loss of elastic modulus to levels of limited use in protein mechanisms, consideration of regular spiral-like structures rather than ramdom chain networks or random coils are proposed for mechanochemical coupling of the second kind. Chemical processes to effect mechanochemical coupling in biological systems are most obviously phosphorylation-dephosphorylation and changes in calcium ion activity but also changes in pH. These issues are considered in the events attending parturition in muscle contraction and in cell motility.     

Comparative and functional morphology of wing coupling structures in Trichoptera: Annulipalpia

Several orders of morphologically four‐winged insects have evolved mechanisms that enforce a union between the mesothoracic and metathoracic wings (forewings and hindwings) during the wing beat cycle. Such mechanisms result in a morphologically tetrapterous insect flying as if it were functionally dipterous, and these mechanisms have been described for several insect orders. The caddisfly suborders Annulipalpia and Integripalpia (Trichoptera) each have evolved a wing coupling apparatus, with at least three systems having evolved within the suborder Annulipalpia. The comparative and inferred functional morphology of the putative wing coupling mechanisms is described for the annulipalpian families Hydropsychidae (subfamilies Macronematinae and Hydropsychinae), Polycentropodidae and Ecnomidae, and a novel form‐functional complex putatively involved with at‐rest forewing‐forewing coupling is described for Hydropsychidae: Smicrideinae. It is proposed that the morphology of the wing coupling apparatuses of Hydropsychinae and Macronematinae are apomorphies for those clades. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Pyridostigmine improves cardiac function and rhythmicity through RyR2 stabilization and inhibition of STIM1-mediated calcium entry in heart failure

Heart failure (HF) is characterized by asymmetrical autonomic balance. Treatments to restore parasympathetic activity in human heart failure trials have shown beneficial effects. However, mechanisms of parasympathetic-mediated improvement in cardiac function remain unclear. The present study examined the effects and underpinning mechanisms of chronic treatment with the cholinesterase inhibitor, pyridostigmine (PYR), in pressure overload HF induced by transverse aortic constriction (TAC) in mice. TAC mice exhibited characteristic adverse structural (left ventricular hypertrophy) and functional remodelling (reduced ejection fraction, altered myocyte calcium (Ca) handling, increased arrhythmogenesis) with enhanced predisposition to arrhythmogenic aberrant sarcoplasmic reticulum (SR) Ca release, cardiac ryanodine receptor (RyR2) hyper-phosphorylation and up-regulated store-operated Ca entry (SOCE). PYR treatment resulted in improved cardiac contractile performance and rhythmic activity relative to untreated TAC mice. Chronic PYR treatment inhibited altered intracellular Ca handling by alleviating aberrant Ca release and diminishing pathologically enhanced SOCE in TAC myocytes. At the molecular level, these PYR-induced changes in Ca handling were associated with reductions of pathologically enhanced phosphorylation of RyR2 serine-2814 and STIM1 expression in HF myocytes. These results suggest that chronic cholinergic augmentation alleviates HF via normalization of both canonical RyR2-mediated SR Ca release and non-canonical hypertrophic Ca signaling via STIM1-dependent SOCE.     

Preparation and properties of an immobilized cellulase on the reversibly soluble matrix Eudragit L-100

Abstract

To prepare a smart biocatalyst, cellulase was immobilized on the reversibly soluble matrix Eudragit L-100 by non-covalent and covalent methods. Covalent immobilization using carbodiimide coupling exhibited superior enzyme loading and reusability compared with non-covalent immobilization, and the covalent loading was increased by almost 20% through the addition of N-hydroxysuccinimide. The temperature optimum of the cellulase was not improved apparently by immobilization but the pH optimum increased from 4.75 to 5.25. Immobilized cellulase was more active than free cellulase above pH 5.0. Immobilized cellulase was more stable than free cellulase during storage at 4°C, room temperature and 50°C. K_m values of immobilized and free cellulase were 85.55 and 73.84 g L^?1, respectively. About 50% productivity was retained after five cycles for hydrolysis of steam-exploded straw.     

Linking of cytochrome P450cam and putidaredoxin by a co-ordination bridge

Cytochrome P450_cam (CYP101) catalyzes the oxidation of D(+)-camphor at the 5 position. The enzyme couples the reduction of dioxygen to the oxidation of the substrate. To transfer electrons from the reductant (NADH) to the cytochrome, two additional proteins are required. These are putidaredoxin (PdX) and putidaredoxin reductase (PdR). We have chemically linked a form of PdX with a histidine tag at the C-terminus to the P450. To accomplish this, we have modified cysteine 334 on P450 with a bipyridinyl group, and co-ordinated the C-terminal histidine tag of PdX by the addition of Ni²⁺ or Ru³⁺. The Ru³⁺ complex was the most stable. The non-linked system gave mostly 5-ketocamphor, a product of two consecutive hydroxylations, and H₂O₂, a product of 2-electron uncoupling. The Ni²⁺ complex gave both 5-exo-hydroxycamphor and 5-ketocamphor, but it also uncoupled. The Ru³⁺ complex gave a single product (5-exo-hydroxycamphor) and did not uncouple at the optimal PdR concentration. Our results are consistent with other studies of this system, in that strong binding of PdX to P450 is crucial for good coupling and for release of 5-exo-hydroxycamphor.     

Control of cardiac contraction by sodium: Promises,reckonings, and new beginnings

Several generations of cardiac physiologists have verified that basal cardiac contractility depends strongly on the transsarcolemmal Na gradient, and the underlying molecular mechanisms that link cardiac excitation-contraction coupling (ECC) to the Na gradient have been elucidated in good detail for more than 30 years. In brief, small increases of cytoplasmic Na push cardiac (NCX1) Na/Ca exchangers to increase contractility by increasing the myocyte Ca load. Accordingly, basal cardiac contractility is expected to be physiologically regulated by pathways that modify the cardiac Na gradient and the function of Na transporters. Assuming that this expectation is correct, it remains to be elucidated how in detail signaling pathways affecting the cardiac Na gradient are controlled in response to changing cardiac output requirements. Some puzzle pieces that may facilitate progress are outlined in this short review. Key open issues include (1) whether the concept of local Na gradients is viable, (2) how in detail Na channels, Na transporters and Na/K pumps are regulated by lipids and metabolic processes, (3) the physiological roles of Na/K pump inactivation, and (4) the possibility that key diffusible signaling molecules remain to be discovered.     

Programmable assembly of 2D crystalline protein arrays into covalently stacked 3D bionanomaterials

Rational embellishment of self-assembling two-dimensional (2D) proteins make it possible to build 3D nanomaterials with novel catalytic, optoelectronic and mechanical properties. However, introducing multiple sites of embellishment into 2D protein arrays without affecting the self-assembly is challenging, limiting the ability to program in additional functionality and new 3D configurations. Here we introduce two orthogonal covalent linkages at multiple sites in a 2D crystalline-forming protein without affecting its self-assembly. We first engineered the surface-layer protein SbsB from Geobacillus stearothermophilus pV72/p2 to display isopeptide bond-forming protein conjugation pairs, SpyTag or SnoopTag, at four positions spaced 5.7-10.5 nm apart laterally and 3 nm axially. The C-terminal and two newly-identified locations within SbsB monomer accommodated the short SpyTag or SnoopTag peptide tags without affecting the 2D lattice structure. Introducing tags at distinct locations enabled orthogonal and covalent binding of SpyCatcher- or SnoopCatcher-protein fusions to micron-sized 2D nanosheets. By introducing different types of bifunctional cross-linkers, the dual-functionalized nanosheets were programmed to self-assemble into different 3D stacks, all of which retain their nanoscale order. Thus, our work creates a modular protein platform that is easy to program to create dual-functionalized 2D and lamellar 3D nanomaterials with new catalytic, optoelectronic, and mechanical properties.