Dimer Formation of a Stabilized Gβ1 Variant: A Structural and Energetic Analysis

In previous work, a strongly stabilized variant of the β1 domain of streptococcal protein G (Gβ1) was obtained by an in vitro selection method. This variant, termed Gβ1-M2, contains the four substitutions E15V, T16L, T18I, and N37L. Here we elucidated the molecular basis of the observed strong stabilizations. The contributions of these four residues were analyzed individually and in various combinations, additional selections with focused Gβ1 gene libraries were performed, and the crystal structure of Gβ1-M2 was determined. All single substitutions (E15V, T16L, T18I, and N37L) stabilize wild-type Gβ1 by contributions of between 1.6 and 6.0 kJ mol^− 1 (at 70 °C). Hydrophobic residues at positions 16 and 37 provide the major contribution to stabilization by enlarging the hydrophobic core of Gβ1. They also increase the tendency to form dimers, as shown by dependence on the concentration of apparent molecular mass in analytical ultracentrifugation, by concentration-dependent stability, and by a strongly increased van't Hoff enthalpy of unfolding. The 0.88-Å crystal structure of Gβ1-M2 and NMR measurements in solution provide the explanation for the observed dimer formation. It involves a head-to-head arrangement of two Gβ1-M2 molecules via six intermolecular hydrogen bonds between the two β strands 2 and 2′ and an adjacent self-complementary hydrophobic surface area, which is created by the T16L and N37L substitutions and a large 120° rotation of the Tyr33 side chain. This removal of hydrophilic groups and the malleability of the created hydrophobic surface provide the basis for the dimer formation of stabilized Gβ1 variants.     

Optimization of medium composition for the production of exopolysaccharides from Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of exopolysaccharides

Optimization of medium composition for the production of exopolysaccharides (EPS) from Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of EPS were carried out. Firstly, the medium components having significant effect on EPS production were screened out to be glucose, yeast extract and diammonium oxalate monohydrate by using a 2⁽⁷⁻³⁾ fractional factorial design. Secondly, the concentrations of the three factors were optimized using central composite design in response surface methodology. As results, a quadratic model was found to fit for EPS production, and the optimal medium composition was determined as following (g/l): 34.12 glucose, 4 peptone, 5.01 yeast extract, 0.88 diammonium oxalate monohydrate, 0.75 MgSO₄ and 1 KH₂PO₄ and 0.0075 thiamine (VB₁). A yield of 2.363 ± 0.04 g/l for EPS was observed in verification experiment. Finally, EPS from P. baumii Pilát was found to have direct immuno-stimulating activity in vitro on splenocyte proliferative response and acid phosphatase activity in peritoneal macrophages in a dose-dependent manner.     

Microsecond rotational motions of eosin-labeled myosin measured by time-resolved anisotropy of absorption and phosphorescence

We have studied submicrosecond and microsecond rotational motions within the contractile protein myosin by observing the time-resolved anisotropy of both absorption and emission from the long-lived triplet state of eosin-5-iodoacetamide covalently bound to a specific site on the myosin head. These results, reporting anisotropy data up to 50 microseconds after excitation, extend by two orders of magnitude the time range of data on time-resolved site-specific probe motion in myosin. Optical and enzymatic analyses of the labeled myosin and its chymotryptic digests show that more than 95% of the probe is specifically attached to sulfhydryl-1 (SH1) on the myosin head. In a solution of labeled subfragment-1 (S-1) at 4 degrees C, absorption anisotropy at 0.1 microseconds after a laser pulse is about 0.27. This anisotropy decays exponentially with a rotational correlation time of 210 ns, in good agreement with the theoretical prediction for end-over-end tumbling of S-1, and with times determined previously by fluorescence and electron paramagnetic resonance. In aqueous glycerol solutions, this correlation time is proportional to viscosity/temperature in the microsecond time range. Furthermore, binding to actin greatly restricts probe motion. Thus the bound eosin is a reliable probe of myosin-head rotational motion in the submicrosecond and microsecond time ranges. Our submicrosecond data for myosin monomers (correlation time 400 ns) also agree with previous results using other techniques, but we also detect a previously unresolvable slower decay component (correlation time 2.6 microseconds), indicating that the faster motions are restricted in amplitude. This restriction is not consistent with the commonly accepted free-swivel model of S-1 attachment in myosin. In synthetic thick filaments of myosin, both fast (700 ns) and slow (5 microseconds) components of anisotropy decay are observed. In contrast to the data for monomers, the anisotropy of filaments has a substantial residual component (26% of the initial anisotropy) that does not decay to zero even at times as long as 50 microseconds, implying significant restriction in overall rotational amplitude. This result is consistent with motion restricted to a cone half-angle of about 50 degrees. The combined results are consistent with a model in which myosin has two principal sites of segmental flexibility, one giving rise to submicrosecond motions (possibly corresponding to the junction between S-1 and S-2) and the other giving rise to microsecond motions (possibly corresponding to the junction between S-2 and light meromyosin).(ABSTRACT TRUNCATED AT 400 WORDS)     

Wave separation versus bandpass filtering: a comparative non-linear analysis of brain α-EEG signals with and without psychotropic drug treatment

Attempts to demonstrate low-dimensional attractor behaviour in the analysis of electroencephalographic (EEG) signals meet with difficulties that in part stem from a departure from single-system dynamics. In order to address this problem, the -waves can be extracted by digital filtering or by wave separation; these two techniques are compared in order to specify the conditions in which finite impulse response (FIR) bandpass filters can be used. The comparison was made using 18 EEG records of 3 min duration under resting conditions (6 subjects, 3 records per subject: prior to apomorphine administration, then 90 min and 150 min post-treatment). No presence of low-dimensional dynamic episodes in -signals was observed without digital processing. Sixty 5 s sections showing attractor behaviour were found after filtering and twenty five 5 s sections after wave separation. The mean correlation dimension was calculated for each experimental condition and for 4 subjects, in order to observe the temporal profile of the drug. When attractors were found after wave separation, bandpass filtering then also showed attractor behaviour, with the same temporal profile. However, the reverse is not true: attractors were found after bandpass filtering that were not present after wave separation; in this case the results deserve confirmation, although the temporal profiles for all cases in which attractors were found after filtering remained comparable.     

The microbiota protects against Pseudomonas aeruginosa pneumonia via γδ T cell-neutrophil axis in mice

Exposure to antimicrobials leading to microbiota dysbiosis has been found to be an independent risk factor for extensively drug-resistant Pseudomonas aeruginosa acquisition. Microbiota dysbiosis may induce imbalanced immune responses and can affect disease susceptibility. However, the potential role of commensal microbiota in bacterial pneumonia is poorly defined. The aim of this study was to investigate the mechanistic basis for the defective host defenses against P. aeruginosa pneumonia induced by antibiotic pretreatment perturbing microbiota. We found that antibiotic pretreatment significantly perturbed the composition of intestinal microbiota. The microbiota dysbiosis impaired host defenses against P. aeruginosa pneumonia, as reflected by the increased bacterial burden and dissemination, compromised local inflammatory responses and shortened survival time in microbiota-depleted mice compared with controls. This impairment correlated with a defective γδ T17 cell and downstream neutrophil responses. Anti-TCRγδ-treated mice had changes similar to those in the microbiota-depleted mice. Overall, our results suggest the importance of microbiota in supporting the host defense against pneumonia, define a crucial role for the γδ T cell-neutrophil axis in the potential mechanism, and delineate the deleterious effects of antibiotic treatment on antibacterial defenses.     

Effect of adrenergic agonists and antagonists on alanine amino transferase,fructose-1:6-bisphosphatase and glucose production in hepatocytes

Using rat hepatocytes we confirmed our previous results that glucagon and -adrenergic agonists increased the enzyme activity of alanine aminotransferase (AAT) and propranolol abolished their effects. Only the enzyme activity was measured and other parameters like quantity of the enzyme or activation due to modification were not looked for. As in perfusion experiment phenylephrine and phenoxybenzamine (-agonist and -antagonist respectively) also increased the AAT activity in isolated rat hepatocytes and propranolol reversed these effects. The additive effect of glucagon and phenoxybenzamine on AAT was also persistant in hepatocyte system.Fructose- 1:6-bisphosphatase (Fru-P₂ase), another key enzyme in gluconeogenic pathway, was elevated by glucagon and other -adrenergic agonists both in liver perfusion and isolated hepatocyte experiments and was brought back to the normal level by propranolol. In this case also only the enzyme activity was measured and no other parameters were looked for. Unlike AAT this enzyme was not stimulated by phenylephrine or phenoxybenzamine. But AAT and Fru-P₂-ase activities were increased significantly by adenylate cyclase activators like fluoride or forskolin. Thus, it appears that the regulation of fru-P₂-ase by glucagon is purely a -receptor mediated process whereas AAT activation shows a mixed type of regulation where some well known -agonist and antagonists are behaving as -agonists.Results further indicate the presence of phosphodiesterase in hepatocyte membrane which was stimulated by glucagon and brought back to the normal level by propranolol.The different adrenergic compounds stated above, not only modified the activity of the above two enzymes but also stimulated glucose production by hepatocytes from alanine which was in turn abolished by propranolol as well as amino oxyacetate (AOA), a highly specified inhibitor of AAT. This confirm the participation of AAT in gluconeogenesis from alanine in liver. Forskolin and fluoride also increased the glucose production from alanine and showed additive effects with glucagon, phenylephrine and phenoxybenzamine.     

Regulation of N-Methyl-d-aspartate receptor-mediated calcium transport and norepinephrine release in rat hippocampus synaptosomes by polyamines

The role of polyamines (PA) synthesis in NMDA receptor-mediated⁴⁵Ca²⁺ fluxes and norepinephrine release was studied in rat hippocampal synaptosomes. NMDA (50M) caused a sharp (>2-fold) transient increase in PA synthesis regulating enzyme, ornithine decarboxylase (ODC) activity with concomitant elevation in PA levels in the order putrescine>spermidine>spermine. ODC inhibitor, -difluoromethylornithine (DFMO), and NMDA antagonist, 2-amino-5-phosphonovaleric acid (D-AP5), both blocked increases in ODC activity and PA levels. Activation of NMDA receptors induced a sharp (3 to 4-fold) and quick (15 seconds) increase in⁴⁵Ca²⁺ uptake by synaptosomes within 15 seconds of exposure at 37°C. The efflux of⁴⁵Ca²⁺ and³H-norepinephrine (NE) release at 22°C from pre-loaded synaptosomes was also significantly (2 to 4-fold) enhanced by NMDA within 15 seconds. These NMDA receptor-mediated effects on calcium fluxes and NE release were blocked by NMDA receptor-antagonists (DAP-5 and MK-801) and PA synthesis inhibitor, DFMO and the DFMO inhibition nullified by exogenous putrescine. These observations establish that ODC/PA cascade play an important role in transduction of excitatory amino acid mediated signals at NMDA receptors.Special issue dedicated to Dr. Sidney Ochs.     

Solution structure of DNA/RNA hybrid duplex with C8-propynyl 2′-deoxyadenosine modifications: Implication of RNase H and DNA/RNA duplex interaction

Solution structures of DNA/RNA hybrid duplexes, d(GCGCA*AA*ACGCG): r(cgcguuuugcg)d(C) (designated PP57), containing two C8-propynyl 2′-deoxyadenosines (A*) and unmodified hybrid (designated U4A4) are solved. The C8-propynyl groups on 2′-deoxyadenosine perturb the local structure of the hybrid duplex, but overall the structure is similar to that of canonical DNA/RNA hybrid duplex except that Hoogsteen hydrogen bondings between A* and U result in lower thermal stability. RNase H is known to cleave RNA only in DNA/RNA hybrid duplexes. Minor groove widths of hybrid duplexes, sugar puckerings of DNA are reported to be responsible for RNase H mediated cleavage, but structural requirements for RNase H mediated cleavage still remain elusive. Despite the presence of bulky propynyl groups of PP57 in the minor groove and greater flexibility, the PP57 is an RNase H substrate. To provide an insight on the interactions between RNase H and substrates we have modeled Bacillus halodurans RNase H-PP57 complex, our NMR structure and modeling study suggest that the residue Gly(15) and Asn(16) of the loop residues between first β sheet and second β sheet of RNase HI of Escherichia coli might participate in substrate binding.     

Effects of different nitrogen forms and combination with foliar spraying with 6-benzylaminopurine on growth,transpiration, and water and potassium uptake and flow in tobacco

NH₄ ⁺-N can have inhibitory effects on plant growth. However, the mechanisms of these inhibitory effects are still poorly understood. In this study, effects of different N forms and a combination of ammonium + 6-benzylaminopurine (6-BA, a synthetic cytokinin) on growth, transpiration, uptake and flow of water and potassium in 88-days-old tobacco (Nicotiana tabacum L. K 326) plants were studied over a period of 12 days. Plants were supplied with equal amounts of N in different forms: NO₃ ^–, NH₄NO₃, NH₄ ⁺ or NH₄ ⁺+6-BA (foliar spraying every 2 days after onset of the treatments). For determining flows and partitioning upper, middle and lower strata of three leaves each were analysed. During the 12 days study period, 50% replacement of NO₃ ^–-N by NH₄ ⁺-N (NH₄NO₃) did not change growth, transpiration, uptake and flow of water and K⁺ compared with the NO₃ ^–-N treatment. However, NH₄ ⁺-N as the sole N-source caused: (i) a substantial decrease in dry weight gain to 42% and 46% of the NO₃ ^–-N and NH₄NO₃ treatments, respectively; (ii) a marked reduction in transpiration rate, due to reduced stomatal conductance, illustrated by more negative leaf carbon-isotope discrimination (¹³C) compared with the NO₃ ^– treatment, especially in upper leaves; (iii) a strong reduction both in total water uptake, and in the rate of water uptake by roots, likely due to a decrease in root hydraulic conductivity; (iv) a marked reduction of K⁺ uptake to 10%. Under NH₄ ⁺ nutrition the middle leaves accumulated 143%, and together with upper leaves 206% and the stem 227% of the K⁺ currently taken up, indicating massive mobilisation of K⁺ from lower leaves and even the roots. Phloem retranslocation of K⁺ from the shoot and cycling through the root contributed 67% to the xylem transport of K⁺, and this was 2.2 times more than concurrent uptake. Foliar 6-BA application could not suppress or reverse the inhibitory effects on growth, transpiration, uptake and flow of water and ions (K⁺) caused by NH₄ ⁺-N treatment, although positive effects by 6-BA application were observed, even when 6-BA (10^–8 M) was supplied in nutrient solution daily with watering. Possible roles of cytokinin to regulate growth and development of NH₄ ⁺-fed plants are discussed.