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Alzheimer’s disease (AD) is the most common neurodegenerative disorder, characterized by cognitive impairment and dementia, resulting from progressive synaptic dysfunction, loss and neuronal cell death. Inclusion body myositis (IBM) is a skeletal muscle degenerative disease, displaying progressive proximal and distal muscle weakness, in association with muscle fiber atrophy, degeneration and death. Studies have shown that the late onset version of AD (LOAD) and sporadic IBM (sIBM) in muscle share many pathological features, including the presence of extracellular plaques of β-amyloid peptides and intracellular tangles of hyperphosphorylated tau proteins. High blood cholesterol is suggested to be a risk factor for LOAD. Many neuropathological changes of LOAD can be reproduced by feeding rabbits a 2% enriched cholesterol diet for 12 weeks. The cholesterol fed rabbit model also simultaneously develops sIBM like pathology, which makes it an ideal model to study the molecular mechanisms common to the development of both diseases. In the present study, we determined the changes of gene expression in rabbit brain and muscle during the progression of LOAD and sIBM pathology using a custom rabbit nucleotide microarray, followed by qRT-PCR analyses. Out of 869 unique transcripts screened, 47 genes showed differential expression between the control and the cholesterol-treated group during the 12 week period and 19 changed transcripts appeared to be common to LOAD and sIBM. The most notable changes are the upregulation of the hemoglobin gene family and the downregulation of the genes required for mitochondrial oxidative phosphorylation in both brain and muscle tissues throughout the time course. The significant overlap on the changes of gene expression in the brain and muscle of rabbits fed with cholesterol-enriched diet supports the notion that LOAD and sIBM may share a common etiology.

Carnation Italian Ringspot virus (CIRV) has evolved a protein called p19 that acts as a suppressor of RNA silencing in the host cell and aids in viral persistence. This protein has been shown to be sensitive to cysteine alkylation resulting in a reduction in its ability to bind to short-interfering RNA (siRNA). To determine the sites within the protein that are sensitive to alkylation, we systematically tested the functional role of each cysteine residue using site-directed mutagenesis. Variants of the p19 protein were created at locations C110, C134 and C160 where the cysteines were replaced by an inert amino acid such as serine or isoleucine. The results from activity measurements of the purified mutant p19 proteins indicate that the mutants maintain the ability to bind siRNAs with nanomolar affinity, however, their stabilities, as measured by circular dichroism (CD), vary. Functional studies in the presence of the cysteine alkylating agent N-ethylmaleimide (NEM) indicated that p19's ability to bind siRNAs and act as a suppressor of RNA silencing is sensitive to alkylation at all three cysteine residues with the maximum effects occurring when C110 and C134 are both alkylated. These results suggest that the role of the cysteine amino acid conservation is likely to preserve the overall structural integrity of p19 for optimal thermostability and subsequent siRNA-binding activity. We find that p19 function is maximally compromised at high levels of thiol alkylation or in an oxidizing environment.

Porcine pancreatic alpha-amylase (PPA) is inhibited by the red kidney bean (Phaseolus vulgaris) inhibitor alpha-AI1 [Eur. J. Biochem. 265 (1999) 20]. Inhibition kinetics were carried out using DP 4900-amylose and maltopentaose as substrate. As shown by graphical and statistical analysis of the kinetic data, the inhibitory mode is of the mixed noncompetitive type whatever the substrate thus involving the EI, EI2, ESI and ESI2 complexes. This contrast with the E2I complex obtained in the crystal and with biophysical studies. Such difference very likely depends on the [I]/[E] ratio. At low ratio, the E2I complex is favoured; at high ratio the EI, ESI and EI2 complexes are formed. The inhibition model also differs from those previously proposed for acarbose [Eur. J. Biochem. 241 (1996) 787 and Eur. J. Biochem. 252 (1998) 100]. In particular, with alpha-AI1, the inhibition takes place only when PPA and alpha-AI are preincubated together before adding the substrate. This indicates that the abortive PPA-alphaAI1 complex is formed during the preincubation period. One additional carbohydrate binding site is also demonstrated yielding the ESI complex. Also, a second protein binding site is found in EI2 and ESI2 abortive complexes. Conformational changes undergone by PPA upon alpha-AI1 binding are shown by higher sensitivity to subtilisin attack. From X-ray analysis of the alpha-AI1-PPA complex (E2I), the major interaction occurs with two hairpin loops L1 (residues 29-46) and L2 (residues 171-189) of alpha-AI1 protruding into the V-shaped active site of PPA. The hydrolysis of alpha-AI1 that accounts for the inhibitory activity is reported.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder characterized by the presence of extracellular plaques of β-amyloid peptides and intracellular tangles of hyperphosphorylated tau proteins in the brain. The vast majority of cases are late onset AD (LOAD), which are genetically heterogeneous and occur sporadically. High blood cholesterol is suggested to be a risk factor for this disease. Several neuropathological changes of LOAD can be reproduced by supplementing a rabbit’s diet with 2% cholesterol for 12 weeks. Accumulating data in the literature suggest that microRNAs (miRNA) participate in the development of AD pathology. The present study focuses on the survey of changes of miRNA expression in rabbit brains during the progression of AD-like pathology using microarray followed by Taq-Man qRT-PCR analyses. Out of 1769 miRNA probes used in the experiments, 99 miRNAs were found to be present in rabbit brain, 57 were newly identified as miRNAs from rabbit brain. Eleven miRNAs showed significant changes over AD-like pathology progression. Among them, the changes of miR-125b, miR-98, miR-107, miR-30, along with 3 members of the let-7 family were similar to those observed in human AD samples, whereas the expression patterns of miR-15a, miR-26b, miR-9 and miR-576-3p were unique to this rabbit LOAD model. The significant up regulation of miR-26b is consistent with the decrease of leptin levels in the brains of cholesterol fed rabbit model for AD, confirming that miR-26b is indeed regulated by leptin and that both leptin and miR-26b may be involved in cholesterol induced AD-like pathology.

alpha-Amylases from the intestinal cavity of two tilapia species, Oreochromis niloticus (ONI-AMY) and Sarotherodon melanotheron (SME-AMY), were purified using ammonium sulfate precipitation, affinity chromatography and chromatofocusing procedures. The purification was approximately 100-fold. The amylolytic activity, specific activity, product distribution, pH and temperature profile of ONI-AMY and SME-AMY are quite similar. The molecular mass differs slightly: 56600 (ONI-AMY) vs. 55500 (SME-AMY). As shown by isoelectric focusing analysis, both amylases contain two isoforms A and B with distinct pI: 7.2 (A) and 7.8 (B), vs. 8.3 (A) and 8.8 (B), respectively. It was not possible to isolate B, since B converts into A with time. The kinetics of the inhibition of ONI-AMY and SME-AMY activity by alpha-, beta- and gamma-cyclodextrin (alpha-, beta- and gamma-CD) were investigated using amylose as the substrate. Statistical analysis of the kinetic data expressed using a general velocity equation and assuming rapid equilibrium showed that the inhibition is of the mixed noncompetitive type. Similar results were obtained with ONI-AMY and SME-AMY. beta- and gamma-CD are stronger inhibitors than alpha-CD. ONI-AMY and SME-AMY are then closely related and show the general features common to the members of the alpha-amylase class (family 13). They enable ONI and SME tilapias to digest starch in food.

The effects of alpha-, beta- and gamma-cyclodextrins on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic alpha-amylase (PPA) were investigated. The results of the statistical analysis performed on the kinetic data using the general initial velocity equation of a one-substrate reaction in the presence of one inhibitor indicate that the type of inhibition involved depends on the substrate used: the inhibition of amylose hydrolysis by alpha-, beta- and gamma-cyclodextrin is of the competitive type, while the inhibition of maltopentaose hydrolysis is of the mixed noncompetitive type. Consistently, the Lineweaver-Burk plots intersect on the vertical axis when amylose is used as the substrate, while in the case of maltopentaose, the intersection occurs at a point located in the second quadrant. The inhibition of the hydrolysis therefore involves only one abortive complex, PPA-cyclodextrin, when amylose is used as the substrate, while two abortive complexes, PPA-cyclodextrin and PPA-maltopentaose-cyclodextrin, are involved with maltopentaose. The mixed noncompetitive inhibition thus shows the existence of one accessory binding site. In any case, only one molecule of inhibitor binds to PPA. In line with these findings, the difference spectra of PPA produced by alpha-, beta- and gamma-cyclodextrin indicate that binding occurs at a tryptophan and a tyrosine residue. The corresponding dissociation constants and the inhibition constants obtained using the kinetic approach are in the same range (1.2-7 mM). The results obtained here on the inhibition of maltopentaose hydrolysis by cyclodextrin are similar to those previously obtained with acarbose as the inhibitor [Alkazaz, M., Desseaux, V., Marchis-Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem. 252, 100-107], but differ from those obtained with amylose as the substrate and acarbose as inhibitor [Alkazaz, M., Desseaux, V., Marchis-Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787-796]. It is concluded that the hydrolysis of both long and short chain substrates requires at least one secondary binding site, including a tryptophan residue.

Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small protein, p19, that acts as a suppressor of RNA silencing. This protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity. Here we demonstrate a new approach for rapidly determining the quantities of siRNA using fluorescence resonance energy transfer (FRET) between the Carnation Italian ringspot virus (CIRV) p19-CFP fusion protein and Cy3-labeled siRNA. The CIRV p19 fusion protein binds double-stranded siRNAs with nanomolar affinity as determined by FRET. [corrected]

Here we report that the phenanthridine derivative covalently linked to a fluorescein moiety (FLEth) can act as a fluorescence based probe for duplex short interfering RNA (siRNA) and that this probe can also be used to report on protein-RNA interactions. A fluorescence resonance energy transfer (FRET) signal that is observed at 600 nm occurs when FLEth is complexed with siRNA. At least 2 molecules of FLEth can bind to 21 nt duplex siRNA, and the dissociation constants for these interactions are reported. We find that FLEth can also report on the interaction of siRNAs with the Carnation Italian ringspot viral suppressor of RNA silencing p19. FLEth does not bind to the siRNA-p19 complex nor can p19 bind to the siRNA-FLEth complex; rather FLEth can report on the fraction of siRNA that is unbound. FLEth can also bind siRNA in delivery systems such as liposomes. Once the siRNA reaches the interior of Huh 7.5 cells, FLEth dissociates from the siRNA and is found in the nucleoli suggesting that FLEth cannot bind to siRNAs that are associated with the RNA silencing machinery.

Eukaryotes have evolved complex cellular responses to double-stranded RNA including the RNA silencing pathway. Tombusviruses have adapted a mechanism to evade RNA silencing that involves a 19 kDa dimeric protein (p19) that is a suppressor of RNA silencing. In order to develop stabilized p19 proteins, linked versions of p19 from the Carnation Italian Ringspot virus (CIRV) were constructed that joined the C-terminus of one subunit to the N-terminus of the second subunit. Like the native CIRV p19, these linked p19 proteins were able to bind to double-stranded siRNAs with nanomolar affinity and discriminate siRNA according to length. In addition, the interdomain linker improved both the stability and binding properties of the p19 dimer. The observed binding properties support the idea that the semi-rigid cross-link favors the folded, binding-competent state of p19. The cross-linked recombinant CIRV-p19s represent novel stabilized suppressors of RNA silencing and may be useful in future biophysical, immunological and cell biology studies.

The cellulosomal family 9 cellulase genes engH, engK, engL, engM, and engY of Clostridium cellulovorans have been cloned and sequenced. We compared the enzyme activity of family 9 cellulosomal cellulases from C. cellulovorans and their derivatives. EngH has the highest activity toward soluble cellulose derivatives such as carboxymethylcellulose (CMC) as well as insoluble cellulose such as acid-swollen cellulose (ASC). EngK has high activity toward insoluble cellulose such as ASC and Avicel. The results of thin-layer chromatography showed that the cleavage products of family 9 cellulases were varied. These results indicated that family 9 endoglucanases possess different modes of attacking substrates and produce varied products. To investigate the functions of the carbohydrate-binding module (CBM) and the catalytic module, truncated derivatives of EngK, EngH, and EngY were constructed and characterized. EngHDeltaCBM and EngYDeltaCBM devoid of the CBM lost activity toward all substrates including CMC. EngKDeltaCBM and EngMDeltaCBM did not lose activity toward CMC but lost activity toward Avicel. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.

Clostridium cellulovorans , an anaerobic bacterium, degrades native substrates efficiently by producing an extracellular enzyme complex called the cellulosome. All cellulosomal enzyme subunits contain dockerin domains that can bind to hydrophobic domains termed cohesins which are repeated nine times in CbpA, the nonenzymatic scaffolding protein of C. cellulovorans cellulosomes. In this study, the synergistic interactions of cellulases (endoglucanase E, EngE; endoglucanase L, EngL) and hemicellulases (arabinofuranosidase A, ArfA; xylanase A, XynA) were determined on the degradation of corn fiber, a natural substrate containing mainly xylan, arabinan, and cellulose. The degradation by XynA and ArfA of cellulose/arabinoxylan was greater than that of corn fiber and resulted in 2.6-fold and 1.4-fold increases in synergy, respectively. Synergistic effects were observed in increments in both simultaneous and sequential reactions with ArfA and XynA. These synergistic enzymes appear to represent potential rate-limiting enzymes for efficient hemicellulose degradation. When mini-cellulosomes were constructed from the cellulosomal enzymes (XynA and EngL) and mini-CbpA with cohesins 1 and 2 (mini-CbpA1&2) and mini-CbpA with cohesins 5 and 6 (mini-CbpA5&6), higher activity was observed than that for the corresponding enzymes alone. Based on the degradation of different types of celluloses and hemicelluloses, the interaction between cellulosomal enzymes (XynA and EngL) and mini-CbpA displayed a diversity that suggests that dockerin-cohesin interaction from C. cellulovorans may be more selective than random.

International audience; The effects of Phaseolus vulgaris inhibitor (alpha-AI) on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic alpha-amylase (PPA) were investigated. Based on a statistical analysis of the kinetic data and using the general velocity equation, which is valid at equilibrium for all types of inhibition in a single-substrate reaction, it was concluded that the inhibitory mode is of the mixed noncompetitive type involving two molecules of inhibitor. In line with this conclusion, the Lineweaver-Burk primary plots intersect in the second quadrant and the secondary plots of the slopes and the intercepts versus the inhibitor concentrations are parabolic curves, whether the substrate used was amylose or maltopentaose. A specific inhibition model of the mixed noncompetitive type applies here. This model differs from those previously proposed for acarbose [Al Kazaz, M., Desseaux, V., Marchis-Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787-796 and Al Kazaz, M., Desseaux, V., Marchis-Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem. 252, 100-107]. In particular, with alpha-AI, the inhibition takes place only when PPA and alpha-AI are preincubated together before the substrate is added. This shows that the inhibitory PPA-alphaAI complex is formed during the preincubation period. Secondly, other inhibitory complexes are formed, in which two molecules of inhibitor are bound to either the free enzyme or the enzyme-substrate complex. The catalytic efficiency was determined both with and without inhibitor. Using the same molar concentration of inhibitor, alpha-AI was found to be a much stronger inhibitor than acarbose. However, when the inhibitor amount is expressed on a weight basis (mg x L-1), the opposite conclusion is drawn. In addition, limited proteolysis was performed on PPA alone and on the alpha-AI-PPA complex. The results show that, in the complex, PPA is more sensitive to subtilisin attack, and shorter fragments are obtained. These data reflect the conformational changes undergone by PPA as the result of the protein inhibitor binding, which differ from those previously observed with acarbose.

It has been brought to the author's attention that the above paper containsmissing text in the Abstract and they would like to correct this. The text was inadvertently deleted during the typesetting and editing of the proofs. On page 166, the Abstract should read as follows: Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small protein, p19, that acts as a suppressor of RNA silencing. This protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity. Here we demonstrate a new approach for rapidly determining the quantities of siRNA using fluorescence resonance energy transfer (FRET) between the Carnation Italian ringspot virus (CIRV) p19-CFP fusion protein and Cy3-labeled siRNA. The CIRV p19 fusion protein binds double-stranded siRNAs with nanomolar affinity as determined by FRET.

The RNA silencing pathway is an important component of the anti-viral immune response in eukaryotes, particularly in plants. In turn, many viruses have evolved mechanisms to evade or suppress this pathway. Tombusviruses such as the Carnation Italian ringspot virus (CIRV) express a 19kDa protein (p19) that is a suppressor of RNA silencing in infected plants. This protein acts as a dimer and binds specifically to short-interfering RNA (siRNA) through electrostatic interactions between charged residues in the binding cleft. Since pH and salt concentrations can vary widely from host to host, we have investigated the influence of these parameters on the siRNA binding activity of CIRV p19. Previously, we established a convenient fluorescence-based method for assaying CIRV p19:siRNA binding using Ni2+–NTA coated 96-well plates. Using this method, we observe that the CIRV p19 protein binds to siRNA with nanomolar affinity and that this binding is sensitive to pH and salt concentration. The pH-dissociation constant profile shows that CIRV p19:siRNA binding is dependent on three different apparent pKa values. The values extrapolated from the curve are 7.1, 8.0 and 10.6 that we interpret as the ionization of one or more histidine, cysteine and lysine residues, respectively. We find that the optimal suppression of RNA silencing by CIRV p19 occurs in the pH range from 6.2 to 7.6.