α<Subscript>1</Subscript>-Thymosin, α<Subscript>2</Subscript>-interferon,and the LKEKK syntetic peptide inhibit the binding of the B subunit of the cholera toxin to intestinal epithelial cell membranes

The ¹²⁵I-labeled B-subunit of the cholera toxin ([125I]CT-B, specific activity of 98 Ci/mmol) was prepared. This subunit was shown to be bound to the membranes which were isolated from epithelial cells of a mucous tunic of the rat thin intestine with high affinity (K _d = 3.7 nM). The binding of the labeled protein was inhibited by the unlabeled α₂-interferon (IFN-α₂), α₁-thymosin, (TM-α₁), and the LKEKK synthetic peptide corresponding to the 16–20 sequence of TM-α₁ and the 131–135 sequence of human IFN-α₂ (Ki 1.0, 1.5, and 2.0 nM, respectively), whereas the KKEKL unlabeled synthetic peptide did not inhibit the binding (K _i > 100 μМ). The LKEKK peptide and CT-B were shown to dose-dependently increase an activity of the soluble guanylate cyclase (sGC) in the concentration range from 10 to 1000 nM. Thus, the binding of TM- α₁, IFN-α₂, and the LKEKK peptide to the CT-B receptor on a surface of the epithelial cells of the mucous tunic of the rat thin intestine resulted in an increase in the intracellular level of cGMP.     

Effects of activation of μ-, κ<Subscript>1</Subscript>-, δ<Subscript>1</Subscript>-, δ<Subscript>2</Subscript>-, and ORL<Subscript>1</Subscript>-receptors on heart resistance to the pathogenic action of delayed ischemia and reperfusion

Different subtypes of opioid receptors (OR) were activated in rats in vivo to study the activation effect on the heart’s resistance to ischemia and reperfusion. It has been established that administration of deltorphin II, a selective δ₂-OR agonist, lowered the infarct size/area at risk index (IS/AAR) by 23%. Naltrexone, naloxone methiodide (an OR inhibitor not penetrating the blood-brain barrier (BBB)), and naltriben (δ₂-antagonist) eliminated the cardioprotective effect of deltorphin II, while BNTX (a δ₁-antagonist) produced no effect on the cardioprotective action of the δ₂-agonist. The infarct-reducing effect of deltorphin II was eliminated by administration of chelerythrine (a protein kinase C (PKC) inhibitor), glibenclamide (a K_ATP-channels inhibitor), and 5-hydroxydecanoate (a mitochondrial K_ATP-channel blocker). Administration of other opioids did not reduce the IS/AAR index. It has been established that all the deltorphins manifest antiarrhythmic potency. Other opioids do not produce any effect on the incidence of arrhythmia occurrences. The antiarrhythmic effect of deltorphin II was eliminated by preliminary administration of naltrexone, naloxone methiodide, and naltriben, but BNTX did not affect the δ₂-agonist’s anti-arrhythmic effect. The preliminary administration of chelerythrine, a PKC inhibitor, eliminated the δ₂ agonist’s antiarrhythmic action. However, glibenclamide and 5-hydroxydecanoate did not alter the antiarrhythmic effect by deltorphin II. Therefore, activation of the peripheral δ₂-ORs reduces the infarct size and prevents the onset of arrhythmias. The antiarrhythmic effect of the δ₂-OR stimulation is mediated by activating PKC and opening the mitochondrial K_ATP-channels. PKC participates in the antiarrhythmic effect of the δ₂-OR activation, but this effect does not depend on the condition of K_ATP-channels.     

Determination of the lifetime of D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack> and HD<Superscript>−</Superscript> ions

A method for determining the lifetime of unstable ions is described. The method is based on measuring the decrease in the ion beam current onto a fixed detector with increasing path length of the ion beam from the ion source to the detector. The measurements performed for D^? ₂ and HD^? molecular ions have shown that their lifetimes are 3.5 ± 0.1 and 4.4 ± 0.1 μs, respectively.     

Analysis of the amide <Superscript>15</Superscript>N chemical shift tensor of the C<Subscript>α</Subscript> tetrasubstituted constituent of membrane-active peptaibols,the α-aminoisobutyric acid residue,compared to those of di- and tri-substituted proteinogenic amino acid residues

In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The analysis of ¹⁵N chemical shift spectra depends on the accuracy of the chemical shift tensors. When investigating alamethicin and other peptaibols, i.e. polypeptides rich in α-aminoisobutyric acid (Aib), the ¹⁵N chemical shift tensor of this C_α-tetrasubstituted amino acid exhibits pronounced differences when compared to glycine, alanine and other proteinogenic residues. Here we present an experimental investigation on the ¹⁵N amide Aib tensor of N-acetyl-Aib-OH and for the Aib residues within peptaibols. Furthermore, a statistical analysis of the tensors published for di- (glycine) and tri-substituted residues has been performed, where for the first time the published data sets are compiled using a common reference. The size of the isotropic chemical shift and main tensor elements follows the order di- < tri- < tetra-substituted amino acids. A ¹⁵N chemical shift-¹H-¹⁵N dipolar coupling correlation NMR spectrum of alamethicin is used to evaluate the consequences of variations in the main tensor elements for the structural analysis of this membrane peptide.     

Effects of carbon nutrition on the physiological expression of HCO<Subscript>3</Subscript><Superscript>–</Superscript> transport and the CO<Subscript>2</Subscript>-concentrating mechanism in the cyanobacterium <Emphasis Type="Italic">Chlorogloeopsis</Emphasis> sp. ATCC 27193

We have examined the effect of inorganic and organic carbon nutrition on the physiological expression of HCO3- transport and the CO2-concentrating mechanism (CCM) in the nutritionally versatile cyanobacterium Chlorogloeopsis sp. ATCC 27193. Cells grown under photoautotrophic conditions in the presence of limiting or replete levels of inorganic carbon (Ci), or grown under mixotrophic (light) or chemoheterotrophic (dark) conditions in the presence of sucrose retained both active CO2 and Na(+)-independent HCO3- transport activity. However, two distinct effects on the kinetic properties of HCO3- transport were observed, which segregated on the basis of phototrophic and chemoheterotrophic growth in the dark. In the former, the apparent substrate affinity of the HCO3- transport system (K0.5) varied (12-fold) in response to the growth Ci or mixotrophy while the maximum rate of HCO3- transport was approximately constant. In the latter case, the K0.5 value was unchanged from the starting value (35 microM) of Ci-limited photoautotrophic cells used to initiate the dark-grown cultures, but transport capacity declined 3-fold. Modulation of the K0.5 (HCO3- transport) value required light. Cellular carboxysome content was unaffected by growth under any of the regimes employed and these structures were the predominant location of ribulose-1,5-bisphosphate carboxylase/oxygenase, as indicated by immunogold electron microscopy. Mixotrophic and chemoheterotrophic growth resulted in a diminished ability to concentrate Ci internally and a reduction in Ci accumulation ratios at low external Ci concentrations. The relationship between photosynthetic carbon fixation and the internal Ci pool varied by 2-fold, with high-Ci-grown cells being the most efficient and mixotrophically grown cells the least, indicating that there was limited capacity to modulate this relationship in response to changes in carbon nutrition. Within broad limits this relationship appeared to be a fixed trait of the strain and an important factor in determining growth rate.     

An alternative flexible conformation of the <Emphasis Type="Italic">E. coli</Emphasis> HUβ<Subscript>2</Subscript> protein: structural,dynamics, and functional aspects

The histone-like HU protein is the major nucleoid-associated protein involved in the dynamics and structure of the bacterial chromosome. Under physiological conditions, the three possible dimeric forms of the E. coli HU protein (EcHUα₂, EcHUβ₂, and EcHUαβ) are in thermal equilibrium between two dimeric conformations (N₂ ↔ I₂) varying in their secondary structure content. High-temperature molecular dynamics simulations combined with NMR experiments provide information about structural and dynamics features at the atomic level for the N₂ to I₂ thermal transition of the EcHUβ₂ homodimer. On the basis of these data, a realistic 3D model is proposed for the major I₂ conformation of EcHUβ₂. This model is in agreement with previous experimental data.     

Assessing the Efficiency of the Ability to Intentionally Variate the Power of β<Subscript>2</Subscript> Frequencies in the Frontal Lobes of the Cerebral Cortex

We performed longitudinal examinations by neurofeedback in 17 subjects. The subjects were trained for 12 training seßsions (three weeks) to voluntarily increase the intensity of the ß₂ frequencies in the frontal EEG electrodes of the right (the D scenario) and the left (the S scenario) hemispheres. All the subjects were divided into three groups depending on the training efficacy: a group of subjects that successfully controlled the ß activity in the frontal electrodes of both hemispheres (nine subjects), a group of subjects that successfully controlled this activity only in the right hemisphere (four subjects), and a group of subjects that failed to train during the specified period (four subjects). Analysis of the obtained data showed that the training efficacy depended on the cognitive activity that was focused on achieving the corresponding EEG effects and on the individual personality characteristics.     

Presence of Ornithine in the Urate-binding α<Subscript>1</Subscript>—<Subscript>2</Subscript>Globulin

THE urate-binding α₁–α₂ globulin has been isolated from human plasma in a highly purified state¹. The protein was purified by DEAE-‘Sephadex’, ammonium sulphate precipitation and semi-preparative Polyacrylamide gel electrophoresis. The urate-binding α₁–α₂ globulin is a rod-shaped glycoprotein, containing 12.1% carbohydrate, with an isoelectric point of 4.6 and a molecular weight of 67,000 ± 4,000. Amino-acid analysis indicated an unknown basic compound which appeared as an extra peak just in front of lysine¹. To identify this compound, high voltage paper electrophoresis has been carried out on a plate electrophoresis apparatus in pyridine-acetate buffer pH 3.5. A spot separated out corresponding to ornithine. Amino-acid analysis on a BC-200 automatic analyser (Bio-Cal Instruments Co., West Germany), with a 54 cm column at 55° C and with 0.35 M sodium citrate buffer, pH 5.28, as elution buffer at a flow-rate of 150 ml./h, showed that ornithine was present. The presence of ornithine in the protein hydrolysate was also verified by gas chromatography/mass spectrometry².     

Influence of Copper,Iron, Zinc and Fe<Stack><Subscript>3</Subscript><Superscript>+</Superscript></Stack> Haemoglobin Levels on the Etiopathogenesis of Chronic Calcific Pancreatitis—A Study in Patients with Pancreatitis

Chronic pancreatitis is a serious condition associated with severe abdominal pain, and a significant percentage of patients progresses to irreversible calcification in pancreas. The present study evaluates the degree to which the levels of trace elements, copper, iron, selenium, zinc and haemoglobin-Fe³⁺, in blood, serum and pancreas have any role to play in the calcification process associated with fibrosis in pancreas. Twenty-seven calcific (CCP) and 23 non-calcific chronic pancreatitis (CP) patients and equal number of age- and sex-matched normal volunteers (50) were enrolled in the study. Surgically removed pancreatic tissue and blood samples were analysed for copper, iron, selenium, zinc, protein, collagen and lipid peroxidation products in terms of malondialdehyde, protein carbonyls, glutathione, methemoglobin, methemoglobin reductase and ceruloplasmin activity levels. We could find that the pancreatic tissue levels of copper, iron, protein and collagen contents were significantly elevated in CCP patients when compared to CP patients. Serum levels of copper, free ionic copper and iron were also elevated in CCP patients. The serum and the pancreatic tissue level of zinc and selenium showed a significant decrease in CCP patients. The level of methemoglobin was elevated more significantly with the concomitant decline in the activity of methemoglobin reductase. There was a positive correlation between the pancreatic level of copper and iron with the collagen and protein levels. The results of the present study revealed that the levels of copper and iron, the pro-oxidants and zinc and selenium may influence calcification process in CCP patients. Hypoxia-related tissue injury due to the formation of oxidised haemoglobin may also contribute to the pathogenesis of calcification in pancreas.     

Estimation of denitrification potential in a karst aquifer using the <Superscript>15</Superscript>N and <Superscript>18</Superscript>O isotopes of NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>

A confined aquifer in the Malm Karst of the Franconian Alb, South Germany was investigated in order to understand the role of the vadose zone in denitrifiaction processes. The concentrations of chemical tracers Sr²⁺ and Cl^– and concentrations of stable isotope ¹⁸O were measured in spring water and precipitation during storm events. Based on these measurements a conceptual model for runoff was constructed. The results indicate that pre-event water, already stored in the system at the beginning of the event, flows downslope on vertical and lateral preferential flow paths. Chemical tracers used in a mixing model for hydrograph separation have shown that the pre-event water contribution is up to 30%. Applying this information to a conceptual runoff generation model, the values of ¹⁵N and ¹⁸O in nitrate could be calculated. Field observations showed the occurence of significant microbial denitrification processes above the soil/bedrock interface before nitrate percolates through to the deeper horizon of the vadose zone. The source of nitrate could be determined and denitrification processes were calculated. Assuming that the nitrate reduction follows a Rayleigh process one could approximate a nitrate input concentration of about 170 mg/l and a residual nitrate concentration of only about 15%. The results of the chemical and isotopic tracers postulate fertilizers as nitrate source with some influence of atmospheric nitrate. The combined application of hydrograph separation and determination of isotope values in ¹⁵N and ¹⁸O of nitrate lead to an improved understanding of microbial processes (nitrification, denitrification) in dynamic systems.     

A computer-aided quantum chemical study of the N<Stack><Subscript>15</Subscript><Superscript>−</Superscript></Stack> cluster

Ab initio (RHF, MP2) and Density Functional Theory (DFT) methods have been used to examine six isomers of the N15m cluster with the 6-31+G* basis set. Different from the known odd-numbered anionic N7m, N9m, and N11m clusters, in which the open-chain structures are the most stable species, the most stable N15m isomer is structure 1 (C1), which may be considered as a complex between the fragments cyclic N5m (D5h) and staggered N10 (D2d). The decomposition pathways of structure 2 (CS), containing two aromatic N5 rings connected by a N5 chain, and the open-chain structure 3 (C2v) were studied at the B3LYP/6-31+G* level of theory. Relative energies were refined at the level of B3LYP/6-311+G(3df,2p)//B3LYP/6-31+G*+ZPE (B3LYP/6-31+G*). The barriers for N2 and N5m (D5h) fission reactions for structure 2 are predicted to be 18.2 and 14.2 kcal x mol(-1), respectively. The corresponding N2+N3m fission barrier for structure 3 is predicted to be 11.2 kcal x mol(-1). Supplementary material is available for this article if you access the article at http://dx.doi.org/10.1007/s00894-003-0118-0. A link in the frame on the left on that page takes you directly to the supplementary material. Figure Structure 1 of the N15m cluster, showing bond distances in A and bond angles in degrees     

The trophic history of Myall Lakes,New South Wales,Australia: interpretations using δ<Superscript>13</Superscript>C and δ<Superscript>15</Superscript>N of the sedimentary record

In an attempt to determine the trophic history of the Myall Lakes complex (New South Wales, Australia) δ¹³C_org, δ¹⁵N and C_org:N profiles were determined for bulk organic matter of two short sediment cores from Bombah Broadwater and Myall Lake. ²¹⁰Pb profiles and sediment types indicate significantly different trophic trajectories during the time periods examined. δ¹³C_org and C_org:N indicate Bombah Broadwater has been dominated by increasing inputs of terrestrial organic material over the last century, thought to be related to watershed disturbance including agricultural activity. Primary production appears to be dominated by phytoplankton. δ¹⁵N remained relatively stable at around 1‰ until the mid–1970s when there was a sharp increase to 4.7‰, interpreted as an influx of sewage-derived material. These observations offer an insight into the recent trophic changes at the site. Sedimentation rates are noticeably lower in Myall Lake and the most recent sediment is a flocculent organic rich deposit overlying mineral clay. δ¹³C_org and C_org:N values indicate a transition from plankton to macrophyte dominated primary production around 1800AD. δ¹⁵N values become increasingly negative from approximately 1900AD. This is interpreted as being due to increasing reliance by macrophytes on nitrogen recycled from decomposing sediments driven by natural infilling and eutrophication in this basin. The contrasting sedimentation rates, sediment types and geochemical profiles suggest the different basins of this water body are subject to substantially different internal and external influences which should be considered in management decisions.     

Cell-free expression,purification, and characterization of the functional β<Subscript>2</Subscript>-adrenergic receptor for multianalyte detection of β-agonists

Large-scale expression of β₂-adrenergic receptor (β₂-AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused β-adrenergic agonists (β-agonists). Cell-based heterologous expression systems have many critical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional β₂-AR in a cell-free system based on Escherichia coli extracts. A codon-optimized porcine β₂-AR gene (codon adaptation index: 0.96) suitable for high expression in E. coli was synthesized and transcribed to the cell-free system, which contributed to increase the expression up to 1.1 mg/ml. After purification using Ni-affinity chromatography, the bioactivity of the purified receptor was measured by novel enzyme-linked receptor assays. It was determined that the relative affinities of the purified β2-AR for β-agonists in descending order were as follows: clenbuterol > salbutamol > ractopamine. Moreover, their IC₅₀ values were 45.99, 60.38, and 78.02 μg/liter, respectively. Although activity of the cell-free system was slightly lower than activity of systems based on insect and mammalian cells, this system should allow production of β2-AR in bulk amounts sufficient for the development of multianalyte screening methods for detecting β-agonist residues.     

<Emphasis Type="Italic">Jeewanu,</Emphasis> or the ‘particles of life’

Starting in the 1960s, the Indian chemist Krishna Bahadur, from the University of Allahabad, published on organic and inorganic particles that he had synthesized and baptized ‘Jeewanu’, or ‘particle of life’. Bahadur conceived of the Jeewanu as a simple form of the living. These studies are presented in a historical perspective and positioned within mid-20th century research on the origin of life, notably the so-called ‘coacervate theory’ of the Soviet biochemist Aleksandr I Oparin. The concepts of life proposed by Bahadur, Oparin and others are discussed from a historical standpoint.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N resonance assignments for the pro-inflammatory cytokine interleukin-36α

Interleukin-36α (IL-36α) is a recently characterised member of the interleukin-1 superfamily. It is involved in the pathogenesis of inflammatory arthritis in one third of psoriasis patients. By binding of IL-36α to its receptor IL-36R via the NF-κB pathway other cytokines involved in inflammatory and apoptotic cascade are activated. The efficacy of complex formation is controlled by N-terminal processing. To obtain a more detailed view on the structure function relationship we performed a heteronuclear multidimensional NMR investigation and here report the ¹H, ¹³C, and ¹⁵N resonance assignments for the backbone and side chain nuclei of the pro-inflammatory cytokine interleukin-36α.