Generation of structured coherent extreme ultraviolet beams from an MgO crystal.

Short wavelength high-harmonic sources are undergoing intense development for applications in spectroscopy and microscopy. Despite recent progress in peak and average power, spatial control over coherent extreme ultraviolet (XUV) beams remains a formidable challenge due to the lack of suitable optical elements for beam shaping and control. Here we demonstrate a robust and precise approach that structures XUV high-order harmonics in space as they are emitted from a nanostructured MgO crystal. Our demonstration paves the way for bridging the numerous applications of shaped light beams from the visible to the short wavelengths, with potential uses for applications in microscopy and nanoscale machining.

Effects of isoquinoline alkaloids from Macleaya cordata on physiological, immunological and inflammatory parameters in healthy beagles: Alkaloids in dog nutrition.

The aim of the study was to investigate nutritional, physiological and immunological effects of a plant-derived blend of isoquinoline alkaloids (Sangrovit® Extra) in healthy dogs. Two groups of healthy, adult beagles (N = 10) were tested in a cross-over experiment, lasting two consecutive three-week periods. The experimental group received 1.2 g additive/kg feed, according to the recommendation of 10-20 mg/kg live weight per day. The control group received the same feed without additive. Complete blood count, immunological parameters and amino acid concentrations in serum were assessed. Faeces were analysed for short-chain fatty acids, lactate and ammonium; moreover, their quantity and consistency were determined. Neither feed intake, total apparent nutrient digestibility (crude protein and fat, organic matter, sodium, potassium) were affected by intake of the product. Lymphocyte and monocyte counts were slightly increased in both groups. Elevation was not treatment dependant. IgA, IgG, haptoglobin in serum and flow cytometric phenotyping of peripheral lymphocytes were not affected by alkaloids supplementation. Numerically greater methionine concentrations in blood serum occurred in the experimental group (p = 0.182). Quantity and consistency of faeces and ammonium concentration in faeces were not affected by the additive. Faecal concentrations of short-chain organic acids differed between groups (acetic acid, % of total SCFA: control group 52.3 ± 5.2 vs. experimental group 57.1 ± 4.5, p = 0.042), lactate concentrations (d-, l- and total) did not. Due to the shift of SCFA proportions in faeces, an effect of isoquinoline alkaloids (IQs) on the metabolic activity of intestinal microbiota is probable. In conclusion, the addition of IQs in the given dose was well tolerated and did not have adverse effects in healthy dogs.

Engineered erythrocytes covalently linked to antigenic peptides can protect against autoimmune disease.

Current therapies for autoimmune diseases rely on traditional immunosuppressive medications that expose patients to an increased risk of opportunistic infections and other complications. Immunoregulatory interventions that act prophylactically or therapeutically to induce antigen-specific tolerance might overcome these obstacles. Here we use the transpeptidase sortase to covalently attach disease-associated autoantigens to genetically engineered and to unmodified red blood cells as a means of inducing antigen-specific tolerance. This approach blunts the contribution to immunity of major subsets of immune effector cells (B cells, CD4+ and CD8+ T cells) in an antigen-specific manner. Transfusion of red blood cells expressing self-antigen epitopes can alleviate and even prevent signs of disease in experimental autoimmune encephalomyelitis, as well as maintain normoglycemia in a mouse model of type 1 diabetes.

Subunit interactions in pig-kidney fructose-1,6-bisphosphatase: binding of substrate induces a second class of site with lowered affinity and catalytic activity.

BACKGROUND: Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P2 and by high concentrations of its substrate Fru-1,6-P2. The mechanism that produces substrate inhibition continues to be obscure. METHODS: Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P2 and Fru-1,6-P2 on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244. RESULTS: The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P2 induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P2, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a "stapler" that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition. CONCLUSIONS: Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits. GENERAL SIGNIFICANCE: Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.

Unraveling multistate unfolding of pig kidney fructose-1,6-bisphosphatase using single tryptophan mutants.

Pig kidney fructose-1,6-bisphosphatase is a homotetrameric enzyme which does not contain tryptophan. In a previous report the guanidine hydrochloride-induced unfolding of the enzyme has been described as a multistate process [Reyes, A. M., Ludwig, H. C., Yañez, A. J., Rodriguez, P. H and Slebe, J. C. (2003) Biochemistry 42, 6956-6964]. To monitor spectroscopically the unfolding transitions, four mutants were constructed containing a single tryptophan residue either near the C1-C2 or the C1-C4 intersubunit interface of the tetramer. The mutants were shown to retain essentially all of the structural and kinetic properties of the enzyme isolated from pig kidney. The enzymatic activity, intrinsic fluorescence, size-exclusion chromatographic profiles and 1-anilinonaphthalene-8-sulfonate binding by the mutants were studied under unfolding equilibrium conditions. The unfolding profiles were multisteps, and formation of hydrophobic structures was detected. The enzymatic activity of wild-type and mutant FBPases as a function of guanidine hydrochloride concentration showed an initial enhancement (maximum approximately 30%) followed by a biphasic decay. The activity and fluorescence results indicate that these transitions involve conformational changes in the fructose-1,6-bisphosphate and AMP domains. The representation of intrinsic fluorescence data as a 'phase diagram' reveals the existence of five intermediates, including two catalytically active intermediates that have not been previously described, and provides the first spectroscopic evidence for the formation of dimers. The intrinsic fluorescence unfolding profiles indicate that the dimers are formed by selective disruption of the C1-C2 interface.

Different involvement for aldolase isoenzymes in kidney glucose metabolism: aldolase B but not aldolase A colocalizes and forms a complex with FBPase.

The expression of aldolase A and B isoenzyme transcripts was confirmed by RT-PCR in rat kidney and their cell distribution was compared with characteristic enzymes of the gluconeogenic and glycolytic metabolic pathway: fructose-1,6-bisphosphatase (FBPase), phosphoenol pyruvate carboxykinase (PEPCK), and pyruvate kinase (PK). We detected aldolase A isoenzyme in the thin limb and collecting ducts of the medulla and in the distal tubules and glomerula of the cortex. The same pattern of distribution was found for PK, but not for aldolase B, PEPCK, and FBPase. In addition, co-localization studies confirmed that aldolase B, FBPase, and PEPCK are expressed in the same proximal cells. This segregated cell distribution of aldolase A and B with key glycolytic and gluconeogenic enzymes, respectively, suggests that these aldolase isoenzymes participate in different metabolic pathways. In order to test if FBPase interacts with aldolase B, FBPase was immobilized on agarose and subjected to binding experiments. The results show that only aldolase B is specifically bound to FBPase and that this interaction was specifically disrupted by 60 microM Fru-1,6-P2. These data indicate the presence of a modulated enzyme-enzyme interaction between FBPase and isoenzyme B. They affirm that in kidney, aldolase B specifically participates, along the gluconeogenic pathway and aldolase A in glycolysis.

Nativelike intermediate on the unfolding pathway of pig kidney fructose-1,6-bisphosphatase.

The unfolding and dissociation of the tetrameric enzyme fructose-1,6-bisphosphatase from pig kidney by guanidine hydrochloride have been investigated at equilibrium by monitoring enzyme activity, ANS binding, intrinsic (tyrosine) protein fluorescence, exposure of thiol groups, fluorescence of extrinsic probes (AEDANS, MIANS), and size-exclusion chromatography. The unfolding is a multistate process involving as the first intermediate a catalytically inactive tetramer. The evidence that indicates the existence of this intermediate is as follows: (1) the loss of enzymatic activity and the concomitant increase of ANS binding, at low concentrations of Gdn.HCl (midpoint at 0.75 M), are both protein concentration independent, and (2) the enzyme remains in a tetrameric state at 0.9 M Gdn.HCl as shown by size-exclusion chromatography. At slightly higher Gdn.HCl concentrations the inactive tetramer dissociates to a compact dimer which is prone to aggregate. Further evidence for dissociation of tetramers to dimers and of dimers to monomers comes from the concentration dependence of AEDANS-labeled enzyme anisotropy data. Above 2.3 M Gdn.HCl the change of AEDANS anisotropy is concentration independent, indicative of monomer unfolding, which also is detected by a red shift of MIANS-labeled enzyme emission. At Gdn.HCl concentrations higher than 3.0 M, the protein elutes from the size-exclusion column as a single peak, with a retention volume smaller than that of the native protein, corresponding to the completely unfolded monomer. In the presence of its cofactor Mg(2+), the denaturated enzyme could be successfully reconstituted into the active enzyme with a yield of approximately 70-90%. Refolding kinetic data indicate that rapid refolding and reassociation of the monomers into a nativelike tetramer and reactivation of the tetramer are sequential events, the latter involving slow and small conformational rearrangements in the refolded enzyme.

Spin currents in magnetic films.

Spin currents injected into magnetic thin films may noticeably change the magnetization of the films. To describe this effect, exchange coupled Landau-Lifshitz equations for the local magnetization and the spin-polarized charge carriers are combined with transport equations for charge and spin currents. For steady state transport one obtains two different instability conditions. Both conditions are supported by recent experimental data on current induced magnetization reversal and spin-wave excitations. No second ferromagnetic layer is needed for excitations due to spin transfer.

Purine N7 groups that are crucial to the interaction of Escherichia coli rnase P RNA with tRNA.

We have detected by nucleotide analog interference mapping (NAIM) purine N7 functional groups in Escherichia coli RNase P RNA that are important for tRNA binding under moderate salt conditions (0.1 M Mg2+, 0.1 M NH4+). The majority of identified positions represent highly or universally conserved nucleotides. Our assay system allowed us, for the first time, to identify c7-deaza interference effects at two G residues (G292, G306). Several c7-deazaadenine interference effects (A62, A65, A136, A249, A334, A351) have also been identified in other studies performed at very different salt concentrations, either selecting for substrate binding in the presence of 0.025 M Ca2+ and 1 M NH4+ or self-cleavage of a ptRNA-RNase P RNA conjugate in the presence of 3 M NH4+ or Na+. This indicates that these N7 functional groups play a key role in the structural organization of ribozyme-substrate and -product complexes. We further observed that a c7-deaza modification at A76 of tRNA interferes with tRNA binding to and ptRNA processing by E. coli RNase P RNA. This finding combined with the strong c7-deaza interference at G292 of RNase P RNA supports a model in which substrate and product binding to E. coli RNase P RNA involves the formation of intermolecular base triples (A258-G292-C75 and G291-G259-A76).

Distinct modes of mature and precursor tRNA binding to Escherichia coli RNase P RNA revealed by NAIM analyses.

We have analyzed by nucleotide analog interference mapping (NAIM) pools of precursor or mature tRNA molecules, carrying a low level of Rp-RMPalphaS (R = A, G, I) or Rp-c7-deaza-RMPalphaS (R = A, G) modifications, to identify functional groups that contribute to the specific interaction with and processing efficiency by Escherichia coli RNase P RNA. The majority of interferences were found in the acceptor stem, T arm, and D arm, including the strongest effects observed at positions G19, G53, A58, and G71. In some cases (interferences at G5, G18, and G71), the affected functional groups are candidates for direct contacts with RNase P RNA. Several modifications disrupt intramolecular tertiary contacts known to stabilize the authentic tRNA fold. Such indirect interference effects were informative as well, because they allowed us to compare the structural constraints required for ptRNA processing versus product binding. Our ptRNA processing and mature tRNA binding NAIM analyses revealed overlapping but nonidentical patterns of interference effects, suggesting that substrate binding and cleavage involves binding modes or conformational states distinct from the binding mode of mature tRNA, the product of the reaction.

Guanosine 2-NH2 groups of Escherichia coli RNase P RNA involved in intramolecular tertiary contacts and direct interactions with tRNA.

We have identified by nucleotide analog interference mapping (NAIM) exocyclic NH2 groups of guanosines in RNase P RNA from Escherichia coli that are important for tRNA binding. The majority of affected guanosines represent phylogenetically conserved nucleotides. Several sites of interference could be assigned to direct contacts with the tRNA moiety, whereas others were interpreted as reflecting indirect effects on tRNA binding due to the disruption of tertiary contacts within the catalytic RNA. Our results support the involvement of the 2-NH2 groups of G292/G293 in pairing with C74 and C75 of tRNA CCA-termini, as well as formation of two consecutive base triples involving C75 and A76 of CCA-ends interacting with G292/A258 and G291/G259, respectively. Moreover, we present first biochemical evidence for two tertiary contacts (L18/P8 and L8/P4) within the catalytic RNA, whose formation has been postulated previously on the basis of phylogenetic comparative analyses. The tRNA binding interference data obtained in this and our previous studies are consistent with the formation of a consecutive nucleotide triple and quadruple between the tetraloop L18 and helix P8. Formation of the nucleotide triple (G316 and A94:U104 in wild-type E. coli RNase P RNA) is also supported by mutational analysis. For the mutant RNase P RNA carrying a G94:C104 double mutation, an additional G316-to-A mutation resulted in a restoration of binding affinity for mature and precursor tRNA.

The inflammatory response system and the availability of plasma tryptophan in patients with primary sleep disorders and major depression.

BACKGROUND: It is now well established that major depression is accompanied by an immune-inflammatory system response and that indicators of the latter are inversely correlated with lower availability of plasma tryptophan in depression. Inflammation and infection can alter sleep architecture, whereas sleep disturbances can impair immune functions. AIMS AND METHODS: The aims of the present study were to examine: (i) immune-inflammatory markers, i.e. serum interleukin-6 (IL-6), IL-8, IL-6 receptor (IL-6R), IL-1R antagonist (IL-1RA), gp130, and prostaglandin E2 (PGE2) production by mitogen-stimulated whole blood and the availability of plasma tryptophan in patients with primary sleep disorders, major depression and healthy volunteers; and (ii) the relationships between the availability of tryptophan and indicators of the immune-inflammatory response system. RESULTS: Mitogen-stimulated release of PGE2, and serum IL-6 and IL-8, were significantly increased in both depressed and sleep disordered patients compared to normal controls. Serum IL-1RA was significantly higher in depressed patients than in normal controls. Patients with depression and sleep disorders had a significantly lower availability of tryptophan than normal controls. There were significant and inverse relationships between the availability of plasma tryptophan and serum IL-1RA, IL-6 and IL-8. CONCLUSIONS: The results suggest that (i) there is an activation of the immune-inflammatory response system in primary sleep disorders and depression; and (ii) the decreased availability of plasma tryptophan may be related to the inflammatory system response.

[Early detection of TUR(transurethral resection) syndrome--ethanol measurement in ventilated patients]. / Die Früherkennung des TUR-Syndroms--Ethanolmessung bei beatmeten Patienten.

A major complication of transurethral resection of the prostate (TURP) is the excessive absorption of irrigation solution resulting in hypervolemia and dilutional hyponatremia. Marking the irrigation fluid with ethanol is a method for the early detection of fluid absorption. Currently this method is being used in spontaneously breathing patients undergoing regional anaesthesia. The goal of this study was to determine whether this method is also reliable for patients undergoing general anaesthesia. Fifty-nine patients underwent TURP in either spinal anaesthesia (SPA), or general anaesthesia with semi-open (ITNO) and semi-closed (ITNC) systems. Plasma alcohol concentrations ([Eth]p), exhaled ethanol ([Eth]e), plasma sodium concentration ([Na+]), and central venous pressure (CVP) were measured. The irrigation fluid contained ethanol in an concentration of approx. 1%. We assumed that significant fluid absorption took place when [Eth]p exceeded 0.1/1000. Measurements were performed immediately prior to and during surgery at 10-minute intervals. [Eth]p correlated directly with [Eth]e for both forms of anaesthesia. [Eth]p and [Na+] correlated inversely both for SPA and ITNC. Changes in [Eth]p did not parallel changes in CVP. Clinically relevant episodes of fluid absorption were accompanied by the detection of exhaled ethanol in all groups. We conclude that measuring exhaled ethanol is a minimal invasive monitoring technique that allows the detection of significant fluid absorption in both spontaneously breathing as well as ventilated patients with sufficient sensitivity. The ethanol levels are not predictive of the sodium concentration both in SPA and general anaesthesia. Thus, additional determinations of [Na+] is recommended whenever [Eth]e exceeds 0.2/1000.

[A comparative study of intravenous opioid analgesia. Sufentanil and alfentanil for extracorporeal shock-wave lithotripsy in urologic patients]. / Vergleichende Untersuchungen zur intravenösen Opioidanalgesie. Sufentanil und Alfentanil zur extrakorporalen Stosswellenlithotripsie bei urologischen Patienten.

UNLABELLED: Extracorporeal shock-wave lithotripsy (ESWL) is the method of choice for the treatment of solitary stones in the kidney or ureter. Early lithotripters required prolonged immobility of the patient and caused considerable pain, necessitating general or epidural anaesthesia during the procedure. Modern lithotripters are quicker, but still require analgesia. Intravenous opioids are currently the drugs in favour. The opioids most commonly used are fentanyl and its shorter-acting analogue, alfentanil. The latter has a more rapid onset and, because of its reduced lipid solubility, is less cumulative. Sufentanil is a new opioid that is also of the phenylpiperidone group and has been recently licensed and introduced in Germany. Its pharmacokinetic and pharmacodynamic properties suggest an intermediate duration of action, high analgesic potency, and cardiovascular stability with diminished respiratory depression. In this prospective double-blind study, the effects of alfentanil and sufentanil on cardiovascular and respiratory parameters, the quality of analgesia, degree of sedation and the number and type of side-effects were compared. PATIENTS AND METHODS: After giving informed consent and with the approval of the hospital ethics committee, 62 patients (ASA I or II) were investigated. They were randomly allocated to two groups, either receiving sufentanil (n = 32) or alfentanil (n = 30) during ESWL. No premedication was given. Excluded were patients with pain prior to treatment, patients treated with a spasmolytic or analgesic drug and those who had undergone ESWL within the last 6 months. The loading dose was given as a 5-min infusion to each group. The heart rate, systolic and diastolic blood pressure, percutaneous oxygen saturation (SpO2), and the transcutaneous capillary carbon dioxide tension (PicCO2) were recorded prior to the procedure (i.e. before administration of opioid), after 1000 and after 2000 shock waves and then 1 and 2 h after the end of lithotripsy. After 1000 and 2000 shock waves, and 1 an 2 h after the treatment, the patients were asked to express their degree of tiredness and pain on a visual analogue scale (VAS). The occurrence of side-effects such as nausea, vomiting, pruritus or other unpleasant sensations were noted by an anaesthesia nurse. Simultaneously, the anaesthetist recorded his/her impression of the patient's tiredness and degree of pain, both by using the VAS. He was not allowed to question the patient, nor was he aware of the patient's own scores. At the end of the observation period both the patient and the anaesthetist related their overall satisfaction with the anaesthetic procedure, again by using the VAS. Data were analysed with the Mann-Whitney-U for comparisons between groups and with the Wilcoxon test within each group. The side-effects were analysed with the Chi-square test. RESULTS: The systolic and diastolic blood pressure remained stable in both groups during and after treatment. The mean heart rate was different between the two groups at the beginning, and after the end of the treatment it dropped in both groups, but no significant difference was seen between groups. The PicCO2 rose from an initial mean of 36.8 mm Hg to a maximum of 44.6 mm Hg after 1000 shock waves in the sufentanil group, and from 37.8 mm Hg to 46.0 mm Hg after 2000 shock waves in the alfentanil group. The differences were significant within groups until 1 h after the end of the treatment, but there was no significant difference between groups. The oxygen saturation SpO2 dropped slightly in both groups. The differences were not significant between groups. In the alfentanil group, one patient had a maximum carbon dioxide tension of 83 mm Hg after 2000 shock waves, whereas in the sufentanil treated group the oxygen saturation fell to 72% in one case. (ABSTRACT TRUNCATED)

Characterisation of the solution conformation of a cyclic RGD peptide analogue by NMR spectroscopy allied with a genetic algorithm approach and constrained molecular dynamics.

The solution conformation of a cyclic RGD peptide analogue, cyclo-(S,S)-2-mercaptobenzoate-arginine-glycine-aspartate-2-mer captoanilide, has been determined via two independent approaches for the searching of conformational space and identification of conformations consistent with NMR and CD spectroscopic data: (i) the use of a binary genetic algorithm and (ii) a molecular dynamics simulation. Inter-proton distances were obtained via analysis of cross-peak volumes from a two-dimensional ROESY NMR spectroscopy experiment at 600 MHz and were used as constraints for the computational calculations. The mercaptoanilide amide proton resonance chemical shift had a very small temperature coefficient, indicating that this proton was hydrogen-bonded. Circular dichroism data showed that, in solution, the torsion angle about the disulfide bond was negative, consistent with one of the distinct conformations around this bond in the 200 ps molecular dynamics simulation. The backbone conformations of the structures resulting from the two different approaches were very similar.