Analytical monitoring of alcoholic fermentation using NIR spectroscopy

Alcoholic fermentation under Saccharomyces cerevisiae yeasts is governed largely by glucose uptake, biomass formation, ethanol and glycerin production, and acidification. In this work, PLS calibration models were developed with a view to determining these analytical parameters from near infrared spectra and analytical data provided by the corresponding reference methods. The models were applied to a set of samples obtained from various fermentation processes. The glucose, ethanol, and biomass values predicted by the models exhibited a high correlation with those provided by the reference method.     

Autophosphorylation of cGMP-dependent protein kinase type II

Cyclic nucleotides are shown to stimulate the autophosphorylation of type II cGMP-dependent protein kinase (cGK) on multiple sites. Mass spectrometric based analyses, using a quadrupole time-of-flight-mass spectrometry instrument revealed that cGMP stimulated the in vitro phosphorylation of residues Ser110 and Ser114, and, at a slow rate, of Ser126 and Thr109 or Ser117, all located in the autoinhibitory region. In addition Ser445 was found to be phosphorylated in a cGMP-dependent manner, whereas Ser110 and Ser97 were already prephosphorylated to a large extent in Sf9 cells. cGMP-dependent phosphorylation of cGK II was also demonstrated in intact COS-1 cells and intestinal epithelium. Substitution of most of the potentially autophosphorylated residues for alanines largely abolished the cGMP stimulation of the autophosphorylation. Prolonged autophosphorylation of purified recombinant cGK II in vitro resulted in a 40-50% increase in basal kinase activity, but its maximal cGMP-stimulated activity and the EC50 for cGMP remained unaltered. Mutation of the major phosphorylatable serines 110, 114, and 445 into "phosphorylation-mimicking" glutamates had no effect on the kinetic parameters of cGK II. However, replacing the slowly autophosphorylated residue Ser126 by Glu rendered cGK II constitutively active. These results show that the fast phase of cyclic nucleotide-stimulated autophosphorylation of cGK II has a relatively small feed forward effect on its activity, whereas the secondary phase, presumably involving Ser126 phosphorylation, may generate a constitutively active form of the enzyme.     

Compound-specific isotopic fractionation patterns suggest different carbon metabolisms among Chloroflexus-like bacteria in hot-spring microbial mats

Stable carbon isotope fractionations between dissolved inorganic carbon and lipid biomarkers suggest photoautotrophy by Chloroflexus-like organisms in sulfidic and nonsulfidic Yellowstone hot springs. Where co-occurring, cyanobacteria appear to cross-feed Chloroflexus-like organisms supporting photoheterotrophy as well, although the relatively small 13C fractionation associated with cyanobacterial sugar biosynthesis may sometimes obscure this process.     

Hemodynamic and metabolic effects of the beta-phosphorylated nitroxide 2-diethoxyphosphoryl-2,5,5-trimethylpyrrolidinoxyl during myocardial ischemia and reperfusion

In vitro, the stable six-membered ring nitroxide 2,2,6,6-tetramethyl-1-piperidine-N-oxyl (TEMPO) is known to protect the ischemic and reperfused myocardium through a mechanism likely to involve the limitation of free radical damage. In vivo, TEMPO's high rate of reduction into diamagnetic nonactive compounds could limit its pharmacological use and its potential as an ESR probe in oxymetry studies. Recently, beta-phosphorylated nitrones and pyrrolidines have been reported to protect against myocardial reperfusion injury better than their nonphosphorylated analogs. Using hemodynamic, metabolic, and enzymatic indices of reperfusion injury, the efficacy of 2-diethoxyphosphoryl-2,5,5-trimethylpyrrolidinoxyl (TMPPO), a five-membered ring beta-phosphorylated nitroxide, has been compared to that of TEMPO when added at a nontoxic concentration (1 mM) in buffer-perfused isolated rat hearts during low-flow ischemia, total ischemia, and reflow. TMPPO, which is five times as hydrophilic and eight times as resistant to reduction in a biological medium as TEMPO, was more effective in reducing postischemic contracture and myocardial enzymatic leakage. Since a diamagnetic analog of TMPPO was far less protective and both nitroxides showed an antilipoperoxidant effect and acted mainly when administered only at reflow, it was proposed that beta-phosphorylated nitroxides such as TMPPO could be interesting alternatives in pharmacological and ESR applications.     

Local tissue interactions across the dorsal midline of the forebrain establish CNS laterality

The mechanisms that establish behavioral, cognitive, and neuroanatomical asymmetries are poorly understood. In this study, we analyze the events that regulate development of asymmetric nuclei in the dorsal forebrain. The unilateral parapineal organ has a bilateral origin, and some parapineal precursors migrate across the midline to form this left-sided nucleus. The parapineal subsequently innervates the left habenula, which derives from ventral epithalamic cells adjacent to the parapineal precursors. Ablation of cells in the left ventral epithalamus can reverse laterality in wild-type embryos and impose the direction of CNS asymmetry in embryos in which laterality is usually randomized. Unilateral modulation of Nodal activity by Lefty1 can also impose the direction of CNS laterality in embryos with bilateral expression of Nodal pathway genes. From these data, we propose that laterality is determined by a competitive interaction between the left and right epithalamus and that Nodal signaling biases the outcome of this competition.     

A C-terminal EGF-like domain governs BAD1 localization to the yeast surface and fungal adherence to phagocytes,but is dispensable in immune modulation and pathogenicity of Blastomyces dermatitidis

BAD1, an adhesin and immune modulator of Blastomyces dermatitidis, is an essential virulence factor that is released extracellularly before association with the yeast surface. Here, deletion of the C-terminal EGF-like domain profoundly affected BAD1 function, leading to non-association with yeast, extracellular accumulation and impaired yeast adherence to macrophages. In equilibrium binding assays, DeltaC-term BAD1, lacking an EGF-like domain, bound poorly to BAD1 null yeast, yielding a low affinity (Kd, 3 x 10(-7) M versus 5 x 10(-8) M) and Bmax (1.9 x 10(5) versus 7.9 x 10(5)) compared with BAD1. Similar protein binding profiles were observed using chitin particles, reinforcing the notion that chitin fibrils are a receptor for BAD1, and that the EGF-like domain is critical for BAD1 interactions with chitin on yeast. DeltaC-term strains bound poorly to macrophages, compared with parental or BAD1-reconstituted null strains. However, DeltaC-term strains and the purified protein itself sharply suppressed tumour necrosis factor (TNF)-alpha release by phagocytes in vitro and in lung in vivo, and the strains retained pathogenicity in a murine model of blastomycosis. Our results illustrate the previously undefined role of the EGF-like domain for BAD1 localization to yeast surfaces during cell wall biogenesis. They also demonstrate that the requirements for host cell binding and immune modulation by BAD1 can be dissociated from one another, and that the former is unexpectedly dispensable in the requisite role of BAD1 in pathogenesis.     

Solvation and the hidden thermodynamics of a zinc finger probed by nonstandard repair of a protein crevice

The classical Zn finger contains a phenylalanine at the crux of its three architectural elements: a beta-hairpin, an alpha-helix, and a Zn(2+)-binding site. Surprisingly, phenylalanine is not required for high-affinity Zn2+ binding, but instead contributes to the specification of a precise DNA-binding surface. Substitution of phenylalanine by leucine leads to a floppy but native-like structure whose Zn affinity is maintained by marked entropy-enthalpy compensation (DeltaDeltaH -8.3 kcal/mol and -TDeltaDeltaS 7.7 kcal/mol). Phenylalanine and leucine differ in shape, size, and aromaticity. To distinguish which features correlate with dynamic stability, we have investigated a nonstandard finger containing cyclohexanylalanine at this site. The structure of the nonstandard finger is similar to that of the native domain. The cyclohexanyl ring assumes a chair conformation, and conformational fluctuations characteristic of the leucine variant are damped. Although the nonstandard finger exhibits a lower affinity for Zn2+ than does the native domain (DeltaDeltaG -1.2 kcal/mol), leucine-associated perturbations in enthalpy and entropy are almost completely attenuated (DeltaDeltaH -0.7 kcal/mol and -TDeltaDeltaS -0.5 kcal/mol). Strikingly, global changes in entropy (as inferred from calorimetry) are in each case opposite in sign from changes in configurational entropy (as inferred from NMR). This seeming paradox suggests that enthalpy-entropy compensation is dominated by solvent reorganization rather than nominal molecular properties. Together, these results demonstrate that dynamic and thermodynamic perturbations correlate with formation or repair of a solvated packing defect rather than type of physical interaction (aromatic or aliphatic) within the core.     

Functional consequences of deleting the two C-terminal residues of the scorpion toxin BmTX3

We deleted the two C-terminal residues of the scorpion toxin BmTx3, a peptidyl inhibitor of a transient A-type K(+) current in striatum neurons in culture, to assess their contribution to receptor recognition. The sBmTX3-delYP analog was shown to have a native-like structure in one-dimensional 1H-nuclear magnetic resonance (NMR) spectroscopy. We found that sBmTX3-delYP bound to its receptor less efficiently than the wild-type molecule (by a factor of about 10(5)) in binding assays with rat brain membranes, and that this molecule did not block the A-type K(+) current (at a concentration of 35 microM) in whole-cell patch clamp experiments with striatum neurons. Also, these results show that the A-type K(+) channel blocked by BmTX3 should have a canonical K(+) channel pore structure.     

Involvement of a plastid terminal oxidase in plastoquinone oxidation as evidenced by expression of the Arabidopsis thaliana enzyme in tobacco

Chlororespiration has been defined as a respiratory electron transport chain in interaction with photosynthetic electron transport involving both non-photochemical reduction and oxidation of plastoquinones. Different enzymatic activities, including a plastid-encoded NADH dehydrogenase complex, have been reported to be involved in the non-photochemical reduction of plastoquinones. However, the enzyme responsible for plasquinol oxidation has not yet been clearly identified. In order to determine whether the newly discovered plastid oxidase (PTOX) involved in carotenoid biosynthesis acts as a plastoquinol oxidase in higher plant chloroplasts, the Arabidopsis thaliana PTOX gene (At-PTOX) was expressed in tobacco under the control of a strong constitutive promoter. We showed that At-PTOX is functional in tobacco chloroplasts and strongly accelerates the non-photochemical reoxidation of plastoquinols; this effect was inhibited by propyl gallate, a known inhibitor of PTOX. During the dark to light induction phase of photosynthesis at low irradiances, At-PTOX drives significant electron flow to O(2), thus avoiding over-reduction of plastoquinones, when photo- synthetic CO(2) assimilation was not fully induced. We proposed that PTOX, by modulating the redox state of intersystem electron carriers, may participate in the regulation of cyclic electron flow around photosystem I.