Acetohydroxyacid synthase isozyme I from Escherichia coli has unique catalytic and regulatory properties

AHAS I is an isozyme of acetohydroxyacid synthase which is apparently unique to enterobacteria. It has been known for over 20 years that it has many properties which are quite different from those of the other two enterobacterial AHASs isozymes, as well as from those of "typical" AHASs which are single enzymes in a given organism. These include a unique mechanism for regulation of expression and the absence of a preference for forming acetohydroxybutyrate. We have cloned the two subunits, ilvB and ilvN, of this Escherichia coli isoenzyme and examined the enzymatic properties of the purified holoenzyme and the enzyme reconstituted from purified subunits. Unlike other AHASs, AHAS I demonstrates cooperative feedback inhibition by valine, and the kinetics fit closely to an exclusive binding model. The formation of acetolactate by AHAS I is readily reversible and acetolactate can act as substrate for alternative AHAS I-catalyzed reactions.     

Effects of integrated polysensory recovery treatment on the functions of the central and autonomic nervous systems

The changes in the parameters of P300 cognitive evoked potentials, the psychological and emotional state, and heart rate variability parameters reflecting the characteristics of autonomic regulation during polysensory physiotherapeutic recovery treatment were studied. A single treatment with combined polysensory stimuli resulted in substantial activation of sympathetic regulation of the heart rate, improvement of the emotional state, and an increase in the P300 amplitude in the central frontal areas, which indicates activation of cognitive processes.     

Enamel dictates whole tooth deformation: A finite element model study validated by a metrology method

In order to understand whole tooth behavior under load the biomechanical role of enamel and dentin has to be determined. We approach this question by comparing the deformation pattern and stiffness of intact teeth under load with the deformation pattern and stiffness of the same teeth after the enamel has been mechanically compromised by introducing a defect. FE models of intact human premolars, based on high resolution micro-CT scans, were generated and validated by in vitro electronic speckle pattern interferometry (ESPI) experiments. Once a valid FE model was established, we exploit the flexibility of the FE model to gain more insight into whole tooth function. Results show that the enamel cap is an intrinsically stiff biological structure and its morphology dictates the way a whole tooth will mechanically behave under load. The mechanical properties of the enamel cap were sufficient to mechanically maintain almost its entire stiffness function under load even when a small defect (cavity simulating caries) was introduced into its structure and breached the crown integrity. We conclude that for the most part, that enamel and not dentin dictates the mechanical behavior of the whole tooth.     

Sub‐angstrom modeling of complexes between flexible peptides and globular proteins

A wide range of regulatory processes in the cell are mediated by flexible peptides that fold upon binding to globular proteins. Computational efforts to model these interactions are hindered by the large number of rotatable bonds in flexible peptides relative to typical ligand molecules, and the fact that different peptides assume different backbone conformations within the same binding site. In this study, we present Rosetta FlexPepDock, a novel tool for refining coarse peptide–protein models that allows significant changes in both peptide backbone and side chains. We obtain high resolution models, often of sub‐angstrom backbone quality, over an extensive and general benchmark that is based on a large nonredundant dataset of 89 peptide–protein interactions. Importantly, side chains of known binding motifs are modeled particularly well, typically with atomic accuracy. In addition, our protocol has improved modeling quality for the important application of cross docking to PDZ domains. We anticipate that the ability to create high resolution models for a wide range of peptide–protein complexes will have significant impact on structure‐based functional characterization, controlled manipulation of peptide interactions, and on peptide‐based drug design. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Hyperchlorhidrosis caused by homozygous mutation in CA12, encoding carbonic anhydrase XII

Excessive chloride secretion in sweat (hyperchlorhidrosis), leading to a positive sweat test, is most commonly indicative of cystic fibrosis yet is found also in conjunction with various metabolic, endocrine, and dermatological disorders. There is conflicting evidence regarding the existence of autosomal-recessive hyperchlorhidrosis. We now describe a consanguineous Israeli Bedouin kindred with autosomal-recessive hyperchlohidrosis whose sole symptoms are visible salt precipitates after sweating, a preponderance to hyponatremic dehydration, and poor feeding and slow weight gain at infancy. Through genome-wide linkage analysis, we demonstrate that the phenotype is due to a homozygous mutation in CA12, encoding carbonic anhydrase XII. The mutant (c.427G>A [p.Glu143Lys]) protein showed 71% activity of the wild-type enzyme for catalyzing the CO₂ hydration to bicarbonate and H⁺, and it bound the clinically used sulfonamide inhibitor acetazolamide with high affinity (K_I of 10 nM). Unlike the wild-type enzyme, which is not inhibited by chloride, bromide, or iodide (K_Is of 73–215 mM), the mutant is inhibited in the submicromolar range by these anions (K_Is of 0.37–0.73 mM).     

Multiple pathways leading from the T-cell antigen receptor to the actin cytoskeleton network

Dynamic rearrangements of the actin cytoskeleton, following T-cell antigen receptor (TCR) engagement, provide the structural matrix and flexibility to enable intracellular signal transduction, cellular and subcellular remodeling, and driving effector functions. Recently developed cutting-edge imaging technologies have facilitated the study of TCR signaling and its role in actin-dependent processes. In this review, we describe how TCR signaling cascades induce the activation of actin regulatory proteins and the formation of actin networks, and how actin dynamics is important for T-cell homeostasis, activation, migration, and other effector functions.     

<Emphasis Type="Italic">Thermobifida fusca</Emphasis> exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A

Cellulosomes are efficient cellulose-degradation systems produced by selected anaerobic bacteria. This multi-enzyme complex is assembled from a group of cellulases attached to a protein scaffold termed scaffoldin, mediated by a high-affinity protein–protein interaction between the enzyme-borne dockerin module and the cohesin module of the scaffoldin. The enzymatic complex is attached as a whole to the cellulosic substrate via a cellulose-binding module (CBM) on the scaffoldin subunit. In previous works, we have employed a synthetic biology approach to convert several of the free cellulases of the aerobic bacterium, Thermobifida fusca, into the cellulosomal mode by replacing each of the enzymes’ CBM with a dockerin. Here we show that although family six enzymes are not a part of any known cellulosomal system, the two family six enzymes of the T. fusca system (endoglucanase Cel6A and exoglucanase Cel6B) can be converted to work as cellulosomal enzymes. Indeed, the chimaeric dockerin-containing family six endoglucanase worked well as a cellulosomal enzyme, and proved to be more efficient than the parent enzyme when present in designer cellulosomes. In stark contrast, the chimaeric family six exoglucanase was markedly less efficient than the wild-type enzyme when mixed with other T. fusca cellulases, thus indicating its incompatibility with the cellulosomal mode of action.     

The dopamine metabolite 3-methoxytyramine is a neuromodulator

Dopamine (3-hydroxytyramine) is a well-known catecholamine neurotransmitter involved in multiple physiological functions including movement control. Here we report that the major extracellular metabolite of dopamine, 3-methoxytyramine (3-MT), can induce behavioral effects in a dopamine-independent manner and these effects are partially mediated by the trace amine associated receptor 1 (TAAR1). Unbiased in vivo screening of putative trace amine receptor ligands for potential effects on the movement control revealed that 3-MT infused in the brain is able to induce a complex set of abnormal involuntary movements in mice acutely depleted of dopamine. In normal mice, the central administration of 3-MT caused a temporary mild hyperactivity with a concomitant set of abnormal movements. Furthermore, 3-MT induced significant ERK and CREB phosphorylation in the mouse striatum, signaling events generally related to PKA-mediated cAMP accumulation. In mice lacking TAAR1, both behavioral and signaling effects of 3-MT were partially attenuated, consistent with the ability of 3-MT to activate TAAR1 receptors and cause cAMP accumulation as well as ERK and CREB phosphorylation in cellular assays. Thus, 3-MT is not just an inactive metabolite of DA, but a novel neuromodulator that in certain situations may be involved in movement control. Further characterization of the physiological functions mediated by 3-MT may advance understanding of the pathophysiology and pharmacology of brain disorders involving abnormal dopaminergic transmission, such as Parkinson's disease, dyskinesia and schizophrenia.     

Contribution of a Xylan-Binding Module to the Degradation of a Complex Cellulosic Substrate by Designer Cellulosomes

Conversion of components of the Thermobifida fusca free-enzyme system to the cellulosomal mode using the designer cellulosome approach can be employed to discover the properties and inherent advantages of the cellulosome system. In this article, we describe the conversion of the T. fusca xylanases Xyn11A and Xyn10B and their synergistic interaction in the free state or within designer cellulosome complexes in order to enhance specific degradation of hatched wheat straw as a model for a complex cellulosic substrate. Endoglucanase Cel5A from the same bacterium and its recombinant dockerin-containing chimera were also studied for their combined effect, together with the xylanases, on straw degradation. Synergism was demonstrated when Xyn11A was combined with Xyn10B and/or Cel5A, and ∼1.5-fold activity enhancements were achieved by the designer cellulosome complexes compared to the free wild-type enzymes. These improvements in activity were due to both substrate-targeting and proximity effects among the enzymes contained in the designer cellulosome complexes. The intrinsic cellulose/xylan-binding module (XBM) of Xyn11A appeared to be essential for efficient substrate degradation. Indeed, only designer cellulosomes in which the XBM was maintained as a component of Xyn11A achieved marked enhancement in activity compared to the combination of wild-type enzymes. Moreover, integration of the XBM in designer cellulosomes via a dockerin module (separate from the Xyn11A catalytic module) failed to enhance activity, suggesting a role in orienting the parent xylanase toward its preferred polysaccharide component of the complex wheat straw substrate. The results provide novel mechanistic insight into the synergistic activity of designer cellulosome components on natural plant cell wall substrates.Thermobifida fusca is an aerobic thermophilic soil bacterium with strong cellulolytic activity (52). The T. fusca enzyme system is an extensively studied free cellulase system in which nearly all of the cellulolytic enzymes have been fully characterized, from the individual enzyme sequences to the three-dimensional structures, as well as the biochemical activities of the native and recombinant proteins. The genome sequence has been published (36), and the number and types of carbohydrate-active enzymes produced by the organism are known. This actinomycete produces six different cellulases that have been well studied (29, 31, 32, 50, 52). T. fusca also has the ability to grow on xylan and produces several enzymes involved in xylan degradation, such as xylanases, β-xylosidase, α-l-arabinofuranosidase, and acetylesterases (1, 21).Previous research has suggested that the multienzyme cellulosome complex from Clostridium thermocellum is far more efficient than free cellulase systems that were tested in degrading polysaccharides (33). The cellulosome system is characterized by the strong bimodular interaction between the cohesin and dockerin modules that integrates the various enzymes into the complex (5, 35, 55). Scaffoldin subunits (nonenzymatic protein components) contain the cohesin modules that incorporate the enzymes into the complex via their resident dockerins. The primary scaffoldin subunit also includes a carbohydrate (cellulose)-binding module (CBM) through which the complex recognizes and binds to the cellulosic substrate (42, 46).In order to evaluate the reasons for the apparent advantage of cellulosomes over free enzymes, it is interesting to compare the properties of the best-characterized free-enzyme systems for degradation of polysaccharides with those of the best-studied cellulosome system. We have initiated a program to convert the free-enzyme system of T. fusca into an artificial designer cellulosome (11-13). The designer cellulosome concept is based on the very high affinity (20, 44) and specific interaction (37, 43, 55) between a cohesin and a dockerin module from the same species. Since the various scaffoldin-borne cohesins of a given species essentially show the same specificity of binding for the enzyme-borne dockerins, designer cellulosomes are constructed from recombinant chimeric scaffoldins containing divergent cohesins from different species, for which matching dockerin-containing enzyme hybrids are prepared, as a platform for promoting synergistic action among enzyme components (5). Free cellulases from the T. fusca system were converted to the cellulosomal mode by replacing their native CBM with a desired dockerin module, and in some cases, the resultant “designer cellulosomes” exhibited enhanced synergistic activity on crystalline cellulosic substrates compared to that of the mixture of wild-type enzymes (11).In this study, we incorporated xylanolytic enzymes into designer cellulosomes and investigated their hydrolytic effects on purified xylans and on a native, complex cellulosic substrate (hatched wheat straw). We focused on T. fusca xylanases 11A and 10B (Xyn11A and Xyn10B), which are the most abundant xylanases produced during growth on xylan (34). Xyn11A and Xyn10B function as endoxylanases (28, 34); Xyn11A contains a C-terminal family 2 CBM that binds both cellulose and xylan, whereas Xyn10B lacks a CBM. In some experiments, one of the previously converted (dockerin-containing) T. fusca endoglucanases, f-5A (11), was also introduced into the designer cellulosomes in order to evaluate cooperation between xylanases and cellulases in hydrolysis of a natural substrate. This study contributes primary information concerning a major feature of cellulosomes that had not been suitably addressed in earlier research: although xylanases are integral components of cellulosomes, their synergistic action in the cellulosome mode has yet to be examined experimentally. The xylan-binding CBM (termed XBM for the purposes of this report) was found to contribute to the activity of the parent Xyn11A enzyme.