Suboptimal Substrate Research Articles

Identification of the Thermal Activation Network in Human 15-Lipoxygenase-2: Divergence from Plant Orthologs and Its Relationship to Hydrogen Tunneling Activation Barriers.

The oxidation of polyunsaturated fatty acids by lipoxygenases (LOXs) is initiated by a C-H cleavage step in which the hydrogen atom is transferred quantum mechanically (i.e., via tunneling). In these reactions, protein thermal motions facilitate the conversion of ground-state enzyme-substrate complexes to tunneling-ready configurations and are thus important for transferring energy from the solvent to the active site for the activation of catalysis. In this report, we employed temperature-dependent hydrogen-deuterium exchange mass spectrometry (TDHDX-MS) to identify catalytically linked, thermally activated peptides in a representative animal LOX, human epithelial 15-LOX-2. TDHDX-MS of wild-type 15-LOX-2 was compared to two active site mutations that retain structural stability but have increased activation energies (Ea) of catalysis. The Ea value of one variant, V427L, is implicated to arise from suboptimal substrate positioning by increased active-site side chain rotamer dynamics, as determined by X-ray crystallography and ensemble refinement. The resolved thermal network from the comparative Eas of TDHDX-MS between wild-type and V426A is localized along the front face of the 15-LOX-2 catalytic domain. The network contains a clustering of isoleucine, leucine, and valine side chains within the helical peptides. This thermal network of 15-LOX-2 is different in location, area, and backbone structure compared to a model plant lipoxygenase from soybean that exhibits a low Ea value of catalysis compared to the human ortholog. The presented data provide insights into the divergence of thermally activated protein motions in plant and animal LOXs and their relationships to the enthalpic barriers for facilitating hydrogen tunneling.

Fallen fruit: A backup resource during winter shaping fruit fly communities

Abstract Fallen fruits provide important feeding and breeding substrates for insects such as Drosophilidae and can be a potential trophic reservoir when usual host fruits become scarce. Recently, two invasive fruit fly species, Drosophila suzukii and Chymomyza amoena, have become established in Europe and are expected to alter existing Drosophilidae communities. In this study, carried out between September 2021 and April 2022 in northern France, we aimed to disentangle the relative roles of microclimatic, landscape and local factors driving the diversity of the Drosophilidae community in decaying fruit across seasons. Minimum site temperature during the week preceding sampling and the proportion of rotten fruit tissue had the strongest positive influence on Drosophilidae abundance and species richness. Drosophilidae abundance also increased with urbanisation (portion of building cover) around the sampled trees. Decaying apples were important breeding sites for C. amoena across seasons, but provided a suboptimal substrate for D. suzukii, which was only present in late summer. This study sheds light on the important role of unharvested fallen crop fruit in maintaining the diversity of an insect family that is generally overlooked in field studies. It also emphasises the importance of considering multiple scales and factors when studying the interactions between invasive species, native species and their shared trophic resources. Finally, our data highlight the importance of the Drosophilidae community in recycling agricultural waste.

Upgrading dilute ethanol to odd-chain carboxylic acids by a synthetic co-culture of Anaerotignum neopropionicum and Clostridium kluyveri

BackgroundDilute ethanol streams generated during fermentation of biomass or syngas can be used as feedstocks for the production of higher value products. In this study, we describe a novel synthetic microbial co-culture that can effectively upgrade dilute ethanol streams to odd-chain carboxylic acids (OCCAs), specifically valerate and heptanoate. The co-culture consists of two strict anaerobic microorganisms: Anaerotignum neopropionicum, a propionigenic bacterium that ferments ethanol, and Clostridium kluyveri, well-known for its chain-elongating metabolism. In this co-culture, A. neopropionicum grows on ethanol and CO2 producing propionate and acetate, which are then utilised by C. kluyveri for chain elongation with ethanol as the electron donor.ResultsA co-culture of A. neopropionicum and C. kluyveri was established in serum bottles with 50 mM ethanol, leading to the production of valerate (5.4 ± 0.1 mM) as main product of ethanol-driven chain elongation. In a continuous bioreactor supplied with 3.1 g ethanol L−1 d−1, the co-culture exhibited high ethanol conversion (96.6%) and produced 25% (mol/mol) valerate, with a steady-state concentration of 8.5 mM and a rate of 5.7 mmol L−1 d−1. In addition, up to 6.5 mM heptanoate was produced at a rate of 2.9 mmol L−1 d−1. Batch experiments were also conducted to study the individual growth of the two strains on ethanol. A. neopropionicum showed the highest growth rate when cultured with 50 mM ethanol (μmax = 0.103 ± 0.003 h−1) and tolerated ethanol concentrations of up to 300 mM. Cultivation experiments with C. kluyveri showed that propionate and acetate were used simultaneously for chain elongation. However, growth on propionate alone (50 mM and 100 mM) led to a 1.8-fold reduction in growth rate compared to growth on acetate. Our results also revealed sub-optimal substrate use by C. kluyveri during odd-chain elongation, where excessive ethanol was oxidised to acetate.ConclusionsThis study highlights the potential of synthetic co-cultivation in chain elongation processes to target the production of OCCAs. Furthermore, our findings shed light on to the metabolism of odd-chain elongation by C. kluyveri.

Knockout of ABCC1 in NCI-H441 cells reveals CF to be a suboptimal substrate to study MRP1 activity in organotypic in vitro models

Multidrug resistance-associated protein 1 (MRP1/ABCC1) is an efflux transporter responsible for the extrusion of endogenous substances as well as xenobiotics and their respective metabolites. Its high expression levels in lung tissue imply a key role in pulmonary drug disposition. Moreover, its association with inflammatory lung diseases underline MRP1’s relevance in drug development and precision-medicine. With the aim to develop a tool to better understand MRP1’s role in drug disposition and lung disease, we generated an ABCC1−/− clone based on the human distal lung epithelial cell line NCI-H441 via a targeted CRISPR/Cas9 approach. Successful knockout (KO) of MRP1 was confirmed by qPCR, immunoblot and Sanger sequencing. To assess potential compensatory upregulation of transporters with a similar substrate recognition pattern as MRP1, expression levels of MRP2–9 as well as OAT1–4, 6, 7 and 10 were measured. Functional transporter activity was determined via release studies with two prodrug/substrate pairs, i.e. 5(6)-carboxyfluorescein (CF; formed from its diacetate prodrug) and S-(6-(7-methylpurinyl))glutathione (MPG; formed from its prodrug 6-bromo-7-methylpurine, BMP), respectively. Lastly, transepithelial electrical resistance (TEER) of monolayers of the KO clone were compared with wildtype (WT) NCI-H441 cells. Of eight initially generated clones, the M2 titled clone showed complete absence of mRNA and protein in accordance with the designed genome edit. In transport studies using the substrate CF, however, no differences between the KO clone and WT NCI-H441 cells were observed, whilst no differences in expression of potential compensatory transporters was noted. On the other hand, when using BMP/MPG, the release of MPG was reduced to 11.5% in the KO clone. Based on these results, CF appears to be a suboptimal probe for the study of MRP1 function, particularly in organotypic in vitro and ex vivo models. Our ABCC1−/− NCI-H441 clone further retained the ability to form electrically tight barriers, making it a useful model to study MRP1 function in vitro.

Performing in spite of starvation: How Saccharomyces cerevisiae maintains robust growth when facing famine zones in industrial bioreactors.

In fed-batch operated industrial bioreactors, glucose-limited feeding is commonly applied for optimal control of cell growth and product formation. Still, microbial cells such as yeasts and bacteria are frequently exposed to glucose starvation conditions in poorly mixed zones or far away from the feedstock inlet point. Despite its commonness, studies mimicking related stimuli are still underrepresented in scale-up/scale-down considerations. This may surprise as the transition from glucose limitation to starvation has the potential to provoke regulatory responses with negative consequences for production performance. In order to shed more light, we performed gene-expression analysis of Saccharomyces cerevisiae grown in intermittently fed chemostat cultures to study the effect of limitation-starvation transitions. The resulting glucose concentration gradient was representative for the commercial scale and compelled cells to tolerate about 76 s with sub-optimal substrate supply. Special attention was paid to the adaptation status of the population by discriminating between first time and repeated entry into the starvation regime. Unprepared cells reacted with a transiently reduced growth rate governed by the general stress response. Yeasts adapted to the dynamic environment by increasing internal growth capacities at the cost of rising maintenance demands by 2.7%. Evidence was found that multiple protein kinase A (PKA) and Snf1-mediated regulatory circuits were initiated and ramped down still keeping the cells in an adapted trade-off between growth optimization and down-regulation of stress response. From this finding, primary engineering guidelines are deduced to optimize both the production host's genetic background and the design of scale-down experiments.

Testing the efficiency of artificial spawning nests for pikeperch (Sander lucioperca L.) under natural conditions (Lake Balaton, Hungary)

Pikeperch (Sander lucioperca L.) is an important species for European aquaculture and recreation fisheries. The wide range overexploitation of its stock highlights the importance of supporting wild stocks, within its native range. Our study was therefore addressed to test the effectiveness of different artificial spawning nests in a natural habitat. Eight types of artificial nests, characterized with different shape, size and substrate were tested in six localities of Lake Balaton (Hungary), in order to revel the difference between their effectiveness and usability. Nest occupancy was recorded and number of eggs on each nest was calculated and compared. Artificial spawning nests proved to be highly effective for pikeperch spawning under natural conditions, even on suboptimal bottom substrate conditions. Nest size seems to be an important factor: our results suggest at least 0.5 m2 substrate area may be required.

Structural and biochemical analysis of human ADP-ribosyl-acceptor hydrolase 3 reveals the basis of metal selectivity and different roles for the two magnesium ions

ADP-ribosylation is a reversible and site-specific post-translational modification that regulates a wide array of cellular signaling pathways. Regulation of ADP-ribosylation is vital for maintaining genomic integrity, and uncontrolled accumulation of poly(ADP-ribosyl)ation triggers a poly(ADP-ribose) (PAR)–dependent release of apoptosis-inducing factor from mitochondria, leading to cell death. ADP-ribosyl-acceptor hydrolase 3 (ARH3) cleaves PAR and mono(ADP-ribosyl)ation at serine following DNA damage. ARH3 is also a metalloenzyme with strong metal selectivity. While coordination of two magnesium ions (MgA and MgB) significantly enhances its catalytic efficiency, calcium binding suppresses its function. However, how the coordination of different metal ions affects its catalysis has not been defined. Here, we report a new crystal structure of ARH3 complexed with its product ADP-ribose and calcium. This structure shows that calcium coordination significantly distorts the binuclear metal center of ARH3, which results in decreased binding affinity to ADP-ribose, and suboptimal substrate alignment, leading to impaired hydrolysis of PAR and mono(ADP-ribosyl)ated serines. Furthermore, combined structural and mutational analysis of the metal-coordinating acidic residues revealed that MgA is crucial for optimal substrate positioning for catalysis, whereas MgB plays a key role in substrate binding. Our collective data provide novel insights into the different roles of these metal ions and the basis of metal selectivity of ARH3 and contribute to understanding the dynamic regulation of cellular ADP-ribosylations during the DNA damage response.

Genomic Clustering Facilitates Nuclear Processing of Suboptimal Pri-miRNA Loci

Nuclear processing of most miRNAs is mediated by Microprocessor, comprised of RNase III enzyme Drosha and its cofactor DGCR8. Here, we uncover a hidden layer of Microprocessor regulation via studies of Dicer-independent mir-451, which is clustered with canonical mir-144. Although mir-451 is fully dependent on Drosha/DGCR8, its short stem and small terminal loop render it an intrinsically weak Microprocessor substrate. Thus, it must reside within a cluster for normal biogenesis, although the identity and orientation of its neighbor are flexible. We use DGCR8 tethering assays and operon structure-function assays to demonstrate that local recruitment and transfer of Microprocessor enhances suboptimal substrate processing. This principle applies more broadly since genomic analysis indicates suboptimal canonical miRNAs are enriched in operons, and we validate several of these experimentally. Proximity-based enhancement of suboptimal hairpin processing provides a rationale for genomic retention of certain miRNA operons and may explain preferential evolutionary emergence of miRNA operons.

Engineering chitinolytic activity into a cellulose-active lytic polysaccharide monooxygenase provides insights into substrate specificity

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of recalcitrant polysaccharides such as cellulose and chitin and play an important role in the enzymatic degradation of biomass. Although it is clear that these monocopper enzymes have extended substrate-binding surfaces for interacting with their fibrous substrates, the structural determinants of LPMO substrate specificity remain largely unknown. To gain additional insight into substrate specificity in LPMOs, here we generated a mutant library of a cellulose-active family AA10 LPMO from Streptomyces coelicolor A3(2) (ScLPMO10C, also known as CelS2) having multiple substitutions at five positions on the substrate-binding surface that we identified by sequence comparisons. Screening of this library using a newly-developed MS-based high-throughput assay helped identify multiple enzyme variants that contained four substitutions and exhibited significant chitinolytic activity and a concomitant decrease in cellulolytic activity. The chitin-active variants became more rapidly inactivated during catalysis than a natural chitin-active AA10 LPMO, an observation likely indicative of suboptimal substrate binding leading to autocatalytic oxidative damage of these variants. These results reveal several structural determinants of LPMO substrate specificity and underpin the notion that productive substrate binding by these enzymes is complex, depending on a multitude of amino acids located on the substrate-binding surface.

Diagnostic value of surrogate CSF biomarkers for Creutzfeldt–Jakob disease in the era of RT-QuIC

Prion real-time quaking-induced conversion (RT-QuIC) is emerging as the most potent assay for the in vivo diagnosis of Creutzfeldt-Jakob disease (CJD), but its full application, especially as a screening test, is limited by suboptimal substrate availability, reagent costs, and incomplete assay standardization. Therefore, the search for the most informative cerebrospinal fluid (CSF) surrogate biomarker is still of primary importance. We compared the diagnostic accuracy of CSF protein 14-3-3, measured with both western blot (WB) and enzyme-linked immunosorbent assay (ELISA), total (t)-tau and neurofilament light chain protein (NfL) alone or in combination with RT-QuIC in 212 subjects with rapidly progressive dementia in which we reached a highly probable clinical diagnosis at follow-up or a definite neuropathological diagnosis. T-tau performed best as surrogate CSF biomarker for the diagnosis of CJD (91.3% sensitivity and 78.9% specificity). The 14-3-3 ELISA assay demonstrated a slightly higher diagnostic value compared to the WB analysis (76.9% vs. 72.2%), but both methods performed worse than the t-tau assay. NfL was the most sensitive biomarker for all sCJD subtypes (>95%), including those with low values of t-tau or 14-3-3, but showed the lowest specificity (43.1%). When ELISA-based biomarkers were adopted as screening tests followed by RT-QuIC, t-tau correctly excluded a higher number of non-CJD cases compared to NfL and 14-3-3 ELISA. Our study showed that among the CSF surrogate biomarkers of potential application for the clinical diagnosis of CJD, t-tau performs best either alone or as screening test followed by RT-QuIC as a second-level confirmatory test.

Effect of catheter ablation on pre-existing abnormalities of left atrial systolic, diastolic, and neurohormonal functions in patients with chronic heart failure and atrial fibrillation.

The critical role of the left atrium (LA) in cardiovascular homoeostasis is mediated by its reservoir, conduit, systolic, and neurohormonal functions. Atrial fibrillation is generally a reflection of underlying disease of the LA, especially in patients with heart failure. Disease-related LA remodelling leads to a decline in both atrial contractility and distensibility along with an impairment in the control of neurohormonal systems that regulate intravascular volume. Catheter ablation can lead to further injury to the atrial myocardium, as evidenced by post-procedural troponin release and tissue oedema. The cardiomyocyte loss leads to replacement fibrosis, which may affect up to 30–35% of the LA wall. These alterations further impair atrial force generation and neurohormonal functions; the additional loss of atrial distensibility can lead to a ‘stiff LA syndrome’, and the fibrotic response predisposes to recurrence of the atrial arrhythmia. Although it intends to restore LA systole, catheter ablation often decreases the chamber’s transport functions. This is particularly likely in patients with long-standing atrial fibrillation and pre-existing LA fibrosis, especially those with increased epicardial adipose tissue (e.g. patients with obesity, diabetes and/or heart failure with a preserved ejection fraction). Although the fibrotic LA in these individuals is an ideal substrate for the development of atrial fibrillation, it may be a suboptimal substrate for catheter ablation. Such patients are not likely to experience long-term restoration of sinus rhythm, and catheter ablation has the potential to worsen their haemodynamic and clinical status. Further studies in this vulnerable group of patients are needed.

Methane distribution and oxidation around the Lena Delta in summer 2013

Abstract. The Lena River is one of the largest Russian rivers draining into the Laptev Sea. The predicted increases in global temperatures are expected to cause the permafrost areas surrounding the Lena Delta to melt at increasing rates. This melting will result in high amounts of methane reaching the waters of the Lena and the adjacent Laptev Sea. The only biological sink that can lower methane concentrations within this system is methane oxidation by methanotrophic bacteria. However, the polar estuary of the Lena River, due to its strong fluctuations in salinity and temperature, is a challenging environment for bacteria. We determined the activity and abundance of aerobic methanotrophic bacteria by a tracer method and by the quantitative polymerase chain reaction. We described the methanotrophic population with a molecular fingerprinting method (monooxygenase intergenic spacer analysis), as well as the methane distribution (via a headspace method) and other abiotic parameters, in the Lena Delta in September 2013. The median methane concentrations were 22 nmol L−1 for riverine water (salinity (S) < 5), 19 nmol L−1 for mixed water (5 < S < 20) and 28 nmol L−1 for polar water (S > 20). The Lena River was not the source of methane in surface water, and the methane concentrations of the bottom water were mainly influenced by the methane concentration in surface sediments. However, the bacterial populations of the riverine and polar waters showed similar methane oxidation rates (0.419 and 0.400 nmol L−1 d−1), despite a higher relative abundance of methanotrophs and a higher estimated diversity in the riverine water than in the polar water. The methane turnover times ranged from 167 days in mixed water and 91 days in riverine water to only 36 days in polar water. The environmental parameters influencing the methane oxidation rate and the methanotrophic population also differed between the water masses. We postulate the presence of a riverine methanotrophic population that is limited by sub-optimal temperatures and substrate concentrations and a polar methanotrophic population that is well adapted to the cold and methane-poor polar environment but limited by a lack of nitrogen. The diffusive methane flux into the atmosphere ranged from 4 to 163 µmol m2 d−1 (median 24). The diffusive methane flux accounted for a loss of 8 % of the total methane inventory of the investigated area, whereas the methanotrophic bacteria consumed only 1 % of this methane inventory. Our results underscore the importance of measuring the methane oxidation activities in polar estuaries, and they indicate a population-level differentiation between riverine and polar water methanotrophs.

The Influence of Substrate Hydraulic Conductivity on Plant Water Status of an Ornamental Container Crop Grown in Suboptimal Substrate Water Potentials

Many soilless substrates are inefficient with regard to water (i.e., high porosity and low water holding capacity), which provides an excellent opportunity to increase water efficiency in containerized production. We suggest that increasing hydraulic conductivity in the dry range of substrate moisture content occurring during production can increase water availability, reduce irrigation volume, and produce high quality, marketable crops. Three substrates were engineered using screened pine bark (PB) and amending with either Sphagnum peatmoss or coir to have higher unsaturated hydraulic conductivity between water potentials of −100 and −300 hPa. There was no correlation between substrate unsaturated hydraulic conductivity and saturated hydraulic conductivity (r = 0.04, P = 0.8985). Established Hydrangea arborescens (L.) ‘Annabelle’ plants were grown in the three engineered and a conventional (control) PB substrates exposed to suboptimal irrigation levels (i.e., held at substrate water potentials between −100 and −300 hPa) for 32 days. The plants in the engineered substrates outperformed the control in every growth and morphological metric measured, as well as exhibiting fewer (or no) physiological drought stress indicators (i.e., vigor, growth, plant development, etc.) compared with the control. We observed increased vigor measures in plants grown in substrates with higher unsaturated hydraulic conductivity, as well as greater plant water uptake. The coir increased unsaturated hydraulic conductivity and provided an increased air space when incorporated into coarse bark vs. if peat was incorporated into bark at the same ratio by volume. Increasing PB hydraulic conductivity, through screening bark or amending bark with fibrous materials, in concert with low irrigations can produce marketable, vigorous crops while reducing water consumed and minimizing water wasted in ornamental container production.

Histone H3 Serine 28 Is Essential for Efficient Polycomb-Mediated Gene Repression in Drosophila.

Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28) whose function is unclear. To assess the importance of H3S28, we generated a Drosophila H3 histone mutant with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2, suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, our data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis.

The effect of fine granular sand on pododermatitis in captive greater flamingos (Phoenicopterus roseus)

AbstractPododermatitis is a worldwide health and animal welfare problem in captive flamingos (Phoenicopteridae). Since sub-optimal substrate or flooring has been described as a factor in the development of pododermatitis in poultry and raptors, it is also suspected to play a role in flamingo foot health. Small groups of flamingos were separated from the main group in an indoor enclosure with artificial grass carpet and, in earlier years, concrete flooring, with additional fine granular sand in the water basin for the study year. Feet were evaluated before and after the separation. Judged subjectively, foot lesions had shown a general increase in the indoor enclosure in earlier years. In contrast, lesion severity and prevalence, scored in accordance with a standardised protocol, decreased when fine granular sand was provided. Since flamingos were observed mostly standing on sand and as this represented the major differentiating factor between years, it is concluded that fine granular sand is a favourable substrate to maintain, and one that may even lead to an improvement in flamingo foot health.

Gene expression array testing of FFPE archival breast tumor samples: an optimized protocol for WG-DASL® sample preparation

Archived formalin-fixed, paraffin embedded (FFPE) tissues constitute a vast, well-annotated, but underexploited resource for the molecular study of cancer progression, largely because degradation, chemical modification, and cross-linking, render FFPE RNA a suboptimal substrate for conventional analytical methods. We report here a modified protocol for RNA extraction from FFPE tissues which maximized the success rate (with 100% of samples) in the expression profiling of a set of 60 breast cancer samples on the WG-DASL platform; yielding data of sufficient quality such that in hierarchical clustering (a) 12/12 (100%) replicates correctly identified their respective counterparts, with a high self-correlation (r=0.979), and (b) the overall sample set grouped with high specificity into ER+ (38/40; 95%) and ER- (18/20; 90%) subtypes. These results indicate that a large fraction of decade-old FFPE samples, of diverse institutional origins and processing histories, can yield RNA suitable for gene expression profiling experiments.

Methylation-independent DNA Binding Modulates Specificity of Repressor of Silencing 1 (ROS1) and Facilitates Demethylation in Long Substrates

DNA cytosine methylation is an epigenetic mark that promotes gene silencing and performs critical roles during reproduction and development in both plants and animals. The genomic distribution of DNA methylation is the dynamic outcome of opposing methylation and demethylation processes. In plants, active demethylation occurs through a base excision repair pathway initiated by 5-methycytosine (5-meC) DNA glycosylases of the REPRESSOR OF SILENCING 1 (ROS1)/DEMETER (DME) family. To gain insight into the mechanism by which Arabidopsis ROS1 recognizes and excises 5-meC, we have identified those protein regions that are required for efficient DNA binding and catalysis. We have found that a short N-terminal lysine-rich domain conserved in members of the ROS1/DME family mediates strong methylation-independent binding of ROS1 to DNA and is required for efficient activity on 5-meC.G, but not for T.G processing. Removal of this domain does not significantly affect 5-meC excision from short molecules, but strongly decreases ROS1 activity on long DNA substrates. This region is not required for product binding and is not involved in the distributive behavior of the enzyme on substrates containing multiple 5-meC residues. Altogether, our results suggest that methylation-independent DNA binding allows ROS1 to perform a highly redundant search for efficient excision of a nondamaged, correctly paired base such as 5-meC in long stretches of DNA. These findings may have implications for understanding the evolution of structure and target specificity in DNA glycosylases.

Functional Profiling of Recombinant NS3 Proteases from All Four Serotypes of Dengue Virus Using Tetrapeptide and Octapeptide Substrate Libraries

Regulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1' and P3'. In contrast, the P2' and P4' substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.

Geomorphic controls, riffle substrate quality, and spawning site selection in two semi‐alluvial salmon rivers in the Gaspé Peninsula, Canada

AbstractThe relationships between valley and channel morphology, spawning substrate quality (content of fine sediment < 2 mm) and the selection of spawning sites by Atlantic salmon (Salmo salar) were investigated along 45 km of two semi‐alluvial, valley‐confined rivers in the Gaspé Peninsula, Canada. Linear and logistic regressions confirm that Atlantic salmon prefer spawning at riffles providing good rather than mediocre or poor spawning substrate, as defined by the percentage sand and the Sand Index of Peterson and Metcalfe. However, exceptionally large concentrations of redds were observed on the few riffles located at island heads, with sub‐optimal substrate quality. This observation suggests that, in addition to content of fine material in the substrate, the morphology of spawning reaches may be a significant factor controlling the intensity of inter‐gravel flow through redds and the consequent selection of spawning sites. In the study systems, the quality of spawning substrate was controlled by ‘large‐scale’ geomorphic attributes at the scale of valley segments (1–5 km here): segments located within a wide valley were actively meandering, had higher sinuosity and bank erosion rates, generally lower shear stresses and presented somewhat higher sand content than segments confined by a narrow valley. Although sand contents were significantly higher, laterally unstable segments in wide valleys still harboured good to excellent spawning substrate overall. The study data do not allow the roles of variations in levels of riffle‐zone shear stress to be distinguished from those of cut bank fines input, to explain the observed inter‐segment association between valley width and riffle fines content. Copyright © 2004 John Wiley & Sons, Ltd.

Bioenergetic remodeling during cellular differentiation: changes in cytochrome c oxidase regulation do not affect the metabolic phenotype.

Myogenesis induces mitochondrial proliferation, a decrease in reactive oxygen species (ROS) production, and an increased reliance upon oxidative phosphorylation. While muscles typically possess 20%-40% excess capacity of cytochrome c oxidase (COX), undifferentiated myoblasts have only 5%-20% of the mitochondrial content of myotubes and muscles. We used two muscle lines (C2C12, Sol8) and 3T3-L1 pre-adipocytes to examine if changes in COX regulation or activity with differentiation cause a shift in metabolic phenotype (i.e., more oxidative, less glycolytic, less ROS). COX activity in vivo can be suppressed by its inhibitor, nitric oxide, or sub-optimal substrate (cytochrome c) concentrations. Inhibition of nitric oxide synthase via L-NAME had no effect on the respiration of adherent undifferentiated cells, although it did stimulate respiration of myoblasts in suspension. While cytochrome c content increased during differentiation, there was no correlation with respiratory rate or reliance on oxidative metabolism. There was no correlation between COX specific activity and oxidative metabolism between cell type or in relation to differentiation. These studies show that, despite the very low activities of COX, undifferentiated myoblasts and pre-adipocytes possess a reserve of COX capacity and changes in COX with differentiation do not trigger the shift in metabolic phenotype.