Thermodynamic and kinetic determinants of Thermotoga maritima cold shock protein stability: a structural and dynamic analysis

The cold shock protein (CSP) from hyperthermophile Thermotoga maritima (TmCSP) is only marginally stable (DeltaG(T(opt)) = 0.3 kcal/mol) at 353 K, the optimum environmental temperature (T(opt)) for T. maritima. In comparison, homologous CSPs from E. coli (DeltaG(T(opt)) = 2.2 kcal/mol) and B. subtilis (DeltaG(T(opt)) = 1.5 kcal/mol) are at least five times more stable at 310 K, the T(opt) for the mesophiles. Yet at the room temperature, TmCSP is more stable (DeltaG(T(R)) = 4.7 kcal/mol) than its homologues (DeltaG(T(R)) = 3.0 kcal/mol for E. coli CSP and DeltaG(T(R)) = 2.1 kcal/mol for B. subtilis CSP). This unique observation suggests that kinetic, rather than thermodynamic, barriers toward unfolding might help TmCSP native structure at high temperatures. Consistently, the unfolding rate of TmCSP is considerably slower than its homologues. High temperature (600 K) complete unfolding molecular dynamics (MD) simulations of TmCSP support our hypothesis and reveal an unfolding scheme unique to TmCSP. For all the studied homologues of TmCSP, the unfolding process first starts at the C-terminal region and N-terminal region unfolds in the end. But for TmCSP, both the terminals resist unfolding for consistently longer simulation times and, in the end, unfold simultaneously. In TmCSP, the C-terminal region is better fortified and has better interactions with the N-terminal region due to the charged residues, R2, E47, E49, H61, K63, and E66, being in spatial vicinity. The electrostatic interactions among these residues are unique to TmCSP. Consistently, the room temperature MD simulations show that TmCSP is more rigid at its N- and C-termini as compared to its homologues from E. coli, B. subtilis, and B. caldolyticus.     

Glucose and type 2A protein phosphatase regulate the interaction between catalytic and regulatory subunits of AMP-activated protein kinase

We have expressed in yeast the different subunits of AMP-activated protein kinase (AMPK) and, by using the two-hybrid system, we have found a glucose-regulated interaction between alpha 2 catalytic and gamma 1 regulatory subunits. This regulation was not affected by known regulators of the corresponding yeast orthologue, the SNF1 complex, such as Reg1 or Hxk2, but it was affected by deletion of regulatory subunits of yeast type 2A protein phosphatase (PP2A) complex. We have also found that Tpd3 and PR65 alpha, the corresponding yeast and mammalian A subunits of PP2A, interacted with AMPK alpha 2 both in yeast and mammals, respectively. This interaction occurred only through the regulatory domain of this subunit. These results suggested a direct involvement of PP2A complex in regulating the interaction between AMPK alpha 2 and gamma 1 in a glucose-dependent manner.     

Impact of residue quality on the C and N mineralization of leaf and root residues of three agroforestry species

A laboratory incubation experiment with ¹⁵N labeled root and leaf residues of 3 agroforestry species (Leucaena leucocephala, Dactyladenia barteri and Flemingia macrophylla) was conducted under controlled conditions (25 C) for 56 days to quantify residue C and N mineralization and its relationship with residue quality.No uniform relation was found between the chemical composition of the above and below residues. The leucaena and dactyladenia roots contained more lignin (8 and 26% respectively) and less N (2.0 and 1.0% respectively) than the respective leaves (2 and 13% lignin and 2.9 and 1.4% N, respectively), whereas the differences between the lignin and N contents of the flemingia leaves and roots were not significant (4.6 and 3.0% lignin and 2.63 and 2.68% N, respectively). The leucaena leaves contained more polyphenols than the roots (6.4 and 3.6%), while the polyphenol content of the leaves and roots of the other residues was similar (5.0 and 5.1% for dactyladenia and 4.0 and 3.5% for flemingia).Three patterns of N mineralization could be distinguished. A first pattern, followed by residues producing the highest amounts of CO₂, showed an initial immobilization of soil derived N, followed by a net release of both soil and residue derived N after 7 days of incubation. A second pattern, followed by the flemingia leaf residues which produced intermediate amounts of CO₂ and had an intermediate quality, showed no significant immobilization of soil derived N, and significant mineralization of residue N. A third pattern, followed by both low quality dactyladenia residues, showed a low release of residue derived N and a continued inmobilization of soil derived N.Residue C mineralization was significantly (p<0.05) correlated with the residue lignin content, C-to-N ratio, and polyphenol-to-N ratio. The proportion of residue N mineralized (immobilized) after 56 days of incubation was significantly correlated with the residue N content (p<0.01) and the C-to-N ratio (p<0.05). The relations were quadratic, rather than linear. The ratio of the proportion of residue N mineralized (immobilized) over the proportion of residue C mineralized after 56 days was highly significantly correlated with the lignin content (p<0.01) and C-to-N (p<0.001), lignin-to-N (p<0.01), polyphenol-to-N (p<0.01) and (lignin+polyphenol)-to-N ratios (p<0.01) in a linear way. This indicates that due to the low availability of the residue C, relatively less N is immobilized for the very low quality residues ((lignin+polyphenol)-to-N ratio: 29.7) than for the residues with a relatively higher quality ((lignin+polyphenol)-to-N ratios between 3.3 and 12.5).     

The Protein Kinase CK2 Holoenzyme Structure Supports Proposed Models of Autoregulation and Trans-Autophosphorylation

Eukaryotic protein kinases are typically strictly controlled by second messenger binding, protein/protein interactions, dephosphorylations or similar processes. None of these regulatory mechanisms is known to work for protein kinase CK2 (former name “casein kinase 2”), an acidophilic and constitutively active eukaryotic protein kinase. CK2 predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) complexed to a dimer of non-catalytic subunits (CK2β). One model of CK2 regulation was proposed several times independently by theoretical docking of the first CK2 holoenzyme structure. According to this model, the CK2 holoenzyme forms autoinhibitory aggregates correlated with trans-autophosphorylation and driven by the down-regulatory affinity between an acidic loop of CK2β and the positively charged substrate binding region of CK2α from a neighboring CK2 heterotetramer. Circular trimeric aggregates in which one-half of the CK2α chains show the predicted inhibitory proximity between those regions were detected within the crystal packing of the human CK2 holoenzyme. Here, we present further in vitro support of the “regulation-by-aggregation” model by an alternative crystal form in which CK2 tetramers are arranged as approximately linear aggregates coinciding essentially with the early predictions. In this assembly, the substrate binding region of every CK2α chain is blocked by a CK2β acidic loop from a neighboring tetramer. We found these crystals with CK2^Andante that contains a CK2β variant mutated in a CK2α-contact helix and described to be responsible for a prolonged circadian rhythm in Drosophila. The increased propensity of CK2^Andante to form aggregates with completely blocked active sites may contribute to this phenotype.     

Luminescence enhancement of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase by three amino acid substitutions

To characterize the luminescence properties of nanoKAZ, a 16 amino acid substituted mutant of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase, the effects of each mutated amino acid were investigated by site-specific mutagenesis. All 16 single substituted KAZ mutants were expressed in Escherichia coli cells and their secretory expressions in CHO-K1 cells were also examined using the signal peptide sequence of Gaussia luciferase. Luminescence activity of KAZ was significantly enhanced by single amino acid substitutions at V44I, A54I, or Y138I. Further, the triple mutant KAZ-V44I/A54I/Y138I, named eKAZ, was prepared and these substitutions synergistically enhanced luminescence activity, showing 66-fold higher activity than wild-KAZ and also 7-fold higher activity than nanoKAZ using coelenterazine as a substrate. Substrate specificity of eKAZ for C2- and/or C6-modified coelenterazine analogues was different from that of nanoKAZ, indicating that three amino acid substitutions may be responsible for the substrate recognition of coelenterazine to increase luminescence activity. In contrast, these substitutions did not stimulate protein secretion from CHO-K1 cells, suggesting that the folded-protein structure of KAZ might be different from that of nanoKAZ.     

Temperature and moisture effects on C and N mineralization from surface applied clover residue

A better understanding of the effect of temperature (T) and moisture on soil microbial activity should improve our ability to predict N mineralization from soil organic matter and crop residues. The objective of this study was to evaluate the effects of water potential () and T on C and N mineralization from unamended Cecil loamy sand soil (clayey, kaolinitic, thermic Typic Kanhapludult) and from crimson clover (Trifolium incarnatum L.) residues applied on the soil surface. Cecil soil was packed into acrylic plastic cylinders, adjusted to -5.0, -1.5, -0.03, or -0.003 MPa, treated with clover residues on the surface or left unamended, and incubated at 10, 20, 28, or 35°C for 21 d. Headspace gas samples for CO2 and N2O determinations were taken periodically and NH3 evolved was trapped. Inorganic N in soil and residue extracts was analyzed after 21 d. When increased from -5.0 to -0.003 MPa, total CO2 evolved from unamended soil increased linearly with ln(-), whereas total CO2 evolved from clover residue increased exponentially with . In both cases the effect of was enhanced as T increased. Two-dimensional (T, ) equations were developed to describe these effects. Apparent net mineralized N from the clover residue increased with until it reached a maximum between -0.5 and -0.03 Mpa.     

beta-Endorphin: structure-activity relationships in the guinea pig ileum and opiate receptor binding assays.

The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of β_h-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of β_h-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.     

Bioactive phenolic compounds: production and extraction by solid-state fermentation. A review

Interest in the development of bioprocesses for the production or extraction of bioactive compounds from natural sources has increased in recent years due to the potential applications of these compounds in food, chemical, and pharmaceutical industries. In this context, solid-state fermentation (SSF) has received great attention because this bioprocess has potential to successfully convert inexpensive agro-industrial residues, as well as plants, in a great variety of valuable compounds, including bioactive phenolic compounds. The aim of this review, after presenting general aspects about bioactive compounds and SSF systems, is to focus on the production and extraction of bioactive phenolic compounds from natural sources by SSF. The characteristics of SSF systems and variables that affect the product formation by this process, as well as the variety of substrates and microorganisms that can be used in SSF for the production of bioactive phenolic compounds are reviewed and discussed.     

Evaluation of bioenergy recovery processes treating organic residues from ethanol fermentation process

This study evaluates a two-stage bioprocess for recovering bioenergy in the forms of hydrogen and methane while treating organic residues of ethanol fermentation from tapioca starch. A maximum hydrogen production rate of 0.77 mmol H₂/g VSS/h can be achieved at volumetric loading rate (VLR) of 56 kg COD/m³/day. Batch results indicate that controlling conditions at S₀/X₀ = 12 with X₀ = 4000 mg VSS/L and pH 5.5-6 are important for efficient hydrogen production from fermentation residues. Hydrogen-producing bacteria enriched in the hydrogen bioreactor are likely utilizing lactate and acetate for biohydrogen production from ethanol-fermentation residues. Organic residues remained in the effluent of hydrogen bioreactor can be effectively converted to methane with a rate of 0.37 mmol CH₄/g VSS/h at VLR of 8 kg COD/m³/day. Approximately 90% of COD in ethanol-fermentation residues can be removed and among that 2% and 85.1% of COD can be recovered in the forms of hydrogen and methane, respectively.