Fatty acid profile in erythrocyte membranes and plasma phospholipids affects significantly the extent of inflammatory response to coronary stent implantation.

In coronary heart disease, the treatment of significant stenosis by percutaneous coronary intervention (PCI) with stent implantation elicits local and systemic inflammatory responses. This study was aimed at evaluation of the dynamics of inflammatory response and elucidation of the relationship between the fatty acid profile of red blood cell (RBC) membranes or plasma phospholipids and inflammation after PCI. High-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), serum amyloid A (SAA), malondialdehyde (MDA) and the fatty acid profiles were determined in patients with advanced coronary artery disease undergoing PCI before, 24 h and 48 h after drug-eluting stent implantation (n=36). Patients after PCI exhibited a significant increase in studied markers (hsCRP, IL-6, SAA, MDA). Many significant associations were found between the increase of IL-6, resp. SAA and the amounts of n-6 polyunsaturated fatty acids (namely linoleic, dihomo-gamma-linolenic, docosatetraenoic and docosapentaenoic acid), resp. saturated fatty acids (pentadecanoic, stearic, nonadecanoic) in erythrocyte membranes. The magnitude of the inflammatory response to PCI is related to erythrocyte membrane fatty acid profile, which seems to be a better potential predictor of elevation of inflammatory markers after PCI than plasma phospholipids.

Long-term stability of parameters of antioxidant status in human serum.

The antioxidant status of serum or plasma can be determined using several commercially available assays. Here, four different assays, total antioxidant status (TAS), its second-generation assay (TAS2), biological antioxidant potential (BAP), and enzymatic assay using horseradish peroxidase (EAOC), were applied on human serum samples to test the temperature stability of antioxidants, upon storage of serum for 12 months. The two or three most commonly used temperatures for storage, that is, - 20, - 70 (or - 80), and - 196°C, were selected. The general conclusion is that all assays were stable at the temperatures tested. In addition, there were almost no statistically significant differences between the samples stored at different temperatures. Only the rank order of the EAOC assay was not very good in samples stored at - 20°C. Also three components contributing to the total antioxidant capacity, uric acid, creatinine and bilirubin, showed no statistically significant differences between the temperatures. Therefore, storage at - 20°C is sufficient to maintain a proper assay outcome of most of the total antioxidant assays, although storage at - 70/80°C is to be preferred for longer storage times.

Oxidative stress and metabolic syndrome in obese adults with and without controlled diet restriction.

BACKGROUND: Metabolic syndrome (MetS) represents a collection of markers associated with cardiovascular morbidity and mortality. Due to its high prevalence and steady increase of the occurrence, prevention or management of MetS is of paramount importance. The aim of our study was to evaluate MetS occurrence and extent of oxidative stress by comparing obese adults after diet optimization with untreated controls. MATERIAL AND METHODS: Oxidative stress markers (total amount of free radicals, malondialdehyde, allantoin, alpha1-antiproteinase, GSSG/GSH ratio), total antioxidant capacity and lipid standardized alpha-tocopherol were determined in 40 obese people and 48 healthy controls. The obese people were divided into two group A: obese with restricted energy intake with lowered dietary carbohydrates (n=20) and group B: with the same grade of obesity but without following dietary recommendations (n=20). RESULTS: Group A exhibited lower oxidative stress markers than group B; free radicals (5.18+/-1.68 vs 8.43+/-3.66 mmol/l, p<0.01), GSSG/GSH ratio (11.74+/-5.01 vs 15.38+/-5.93%, p<0.05) and higher antioxidants: lipid standardized alpha-tocopherol (3.70+/-0.51 vs 3.35+/-0.60, p<0.05) and ceruloplasmin (0.24+/-0.08 vs 0.21+/-0.03 g/l, p<0.05), in the course of same grade of obesity. Furthermore MetS occurrence was found significantly lower was in group A. CONCLUSION: The energy intake restriction by 2000 kJ, mainly due to carbohydrate limitations, was associated with decreased oxidative stress and simultaneously increased lipid-standardized alpha-tocopherol and ceruloplasmin in obese people. These changes correlated with diminished MetS occurrence by about 50% (Tab. 3, Ref. 32). Full Text (Free, PDF) www.bmj.sk.

Antioxidant vitamin pool in senior population.

AIM: To compare plasma concentration of alpha-tocopherol and ascorbic acid in healthy seniors (age over 65 years), senior patients with either diabetes mellitus, acute myocardial infarction or dyslipidemia and recommended values of these vitamins. METHODS: Studied groups included 30 patients with diabetes mellitus (DM); 30 patients 1 - 2 weeks after acute myocardial infarction (AMI); 11 patients with lipid metabolism disorder (LD, total cholesterol > 6.2 mM); and control group of 27 healthy persons. RESULTS: Concentration of alpha-tocopherol in DM group was 14.6 +/- 5.3 microM, in AMI group 13.7 +/- 5.6 microM, in LD group 15.9 +/- 5.6 microM and in control group 12.9 +/- 4.1 microM. No statistically significant differences were found. However, comparison of determined values with levels recommended for prevention revealed remarkable low plasma concentration of alpha-tocopherol in the Czech population. Plasma concentration of ascorbic acid in DM group was 47.07 +/- 22.80 microM, in AMI group 33.15 +/- 12.81 microM, in LD group 45.59 +/- 23.02 microM and in control group 43.28 +/- 26.57 microM. No statistically significant differences were found between the controls and individual groups of patients. Plasma concentrations of vitamin C reached the recommended value in all cases except the AMI group, where it was significantly lower. CONCLUSION: Seniors in the Czech population were proved to be significantly short of alpha-tocopherol, minor shortage of vitamin C was found only in group of patients with myocardial infarction.

Antioxidant vitamin levels and glutathione peroxidase activity during ischemia/reperfusion in myocardial infarction.

The consequences of increased oxidative stress, measured as the level of malondialdehyde (MDA) during ischemia/reperfusion, were studied in 48 patients in the acute phase of myocardial infarction (AMI) and a control group (21 blood donors). The serum levels of alpha-tocopherol and beta-carotene were followed. Immediately after the treatment onset the level of alpha-tocopherol started to decrease, reaching a plateau after 24 h. The consumption of beta-carotene was delayed by 90 min. Steady decline was detected during the whole time interval studied (48 h). Glutathione peroxidase (GPx) activity, as a representative of antioxidant enzymes, was estimated in whole blood. The influx of oxygenated blood was accompanied by a stimulation of GPx activity, which reached its maximum at the time of completed reperfusion. When comparing the AMI patients with the control group, the levels of MDA were found significantly increased, which indicates that oxidative stress is already increased during ischemia. Lower antioxidant levels found in the patients might either already be the result of vitamin consumption during ischemia or be a manifestation of their susceptibility to AMI. Monitored consumption of alpha-tocopherol and beta-carotene during reperfusion indicated that in the case of patients, whose level of antioxidant vitamins is below the threshold limit, a further substantial decrease of antioxidant vitamins during reperfusion could enhance the oxidative damage of the myocardium.

Hydrophobic modulation of heme properties in heme protein maquettes.

We have investigated the properties of the two hemes bound to histidine in the H10 positions of the uniquely structured apo form of the heme binding four-helix bundle protein maquette [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2) for the amino acids at positions 6 (I), 13 (F) and 24 (H), respectively. The primary structure of each alpha-helix, alpha-SH, in [I(6)F(13)H(24)](2) is Ac-CGGGEI(6)WKL.H(10)EEF(13)LKK.FEELLKL.H(24)EERLKK.L-CONH(2). In our nomenclature, [I(6)F(13)H(24)] represents the disulfide-bridged di-alpha-helical homodimer of this sequence, i.e., (alpha-SS-alpha), and [I(6)F(13)H(24)](2) represents the dimeric four helix bundle composed of two di-alpha-helical subunits, i.e., (alpha-SS-alpha)(2). We replaced the histidines at positions H24 in [I(6)F(13)H(24)](2) with hydrophobic amino acids incompetent for heme ligation. These maquette variants, [I(6)F(13)I(24)](2), [I(6)F(13)A(24)](2), and [I(6)F(13)F(24)](2), are distinguished from the tetraheme binding parent peptide, [I(6)F(13)H(24)](2), by a reduction in the heme:four-helix bundle stoichiometry from 4:1 to 2:1. Iterative redesign has identified phenylalanine as the optimal amino acid replacement for H24 in the context of apo state conformational specificity. Furthermore, the novel second generation diheme [I(6)F(13)F(24)](2) maquette was related to the first generation diheme [H10A24](2) prototype, [L(6)L(13)A(24)](2) in the present nomenclature, via a sequential path in sequence space to evaluate the effects of conservative hydrophobic amino acid changes on heme properties. Each of the disulfide-linked dipeptides studied was highly helical (>77% as determined from circular dichroism spectroscopy), self-associates in solution to form a dimer (as determined by size exclusion chromatography), is thermodynamically stable (-DeltaG(H)2(O) >18 kcal/mol), and possesses conformational specificity that NMR data indicate can vary from multistructured to single structured. Each peptide binds one heme with a dissociation constant, K(d1) value, tighter than 65 nM forming a series of monoheme maquettes. Addition of a second equivalent of heme results in heme binding with a K(d2) in the range of 35-800 nM forming the diheme maquette state. Single conservative amino acid changes between peptide sequences are responsible for up to 10-fold changes in K(d) values. The equilibrium reduction midpoint potential (E(m7.5)) determined in the monoheme state ranges from -156 to -210 mV vs SHE and in the diheme state ranges from -144 to -288 mV. An observed heme-heme electrostatic interaction (>70 mV) in the diheme state indicates a syn global topology of the di-alpha-helical monomers. The heme affinity and electrochemistry of the three H24 variants studied identify the tight binding sites (K(d1) and K(d2) values <200 nM) having the lower reduction midpoint potentials (E(m7.5) values of -155 and -260 mV) with the H10 bound hemes in the parent tetraheme state of [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2). The results of this study illustrate that conservative hydrophobic amino acid changes near the heme binding site can modulate the E(m) by up to +/-50 mV and the K(d) by an order of magnitude. Furthermore, the effects of multiple single amino acid changes on E(m) and K(d) do not appear to be additive.

Aromatic ring-flipping in supercooled water: implications for NMR-based structural biology of proteins.

We have characterized, for the first time, motional modes of a protein dissolved in supercooled water: the flipping kinetics of phenylalanyl and tyrosinyl rings of the 6 kDa protein BPTI have been investigated by NMR at temperatures between -3 and -16.5 degrees C. At T = -15 degrees C, the ring-flipping rate constants of Tyr 23, Tyr 35, and Phe 45 are smaller than 2 s(-1), i.e., flip-broadening of aromatic NMR lines is reduced beyond detection and averaging of NOEs through ring-flipping is abolished. This allows neat detection of distinct NOE sets for the individual aromatic (1)H spins. In contrast, the rings of Phe 4, Tyr 10, Tyr 21, Phe 22, and Phe 33 are flipping rapidly on the chemical shift time scale with rate constants being in the range from approximately 10(2) to 10(5) s(-1) even at T = -15 degrees C. Line width measurements in 2D [(1)H,(1)H]-NOESY showed that flipping of the Phe 4 and Phe 33 rings is, however, slowed to an extent that the onset of associated line broadening in the fast exchange limit is registered. The reduced ring-flipping rate constant of Phe 45 in supercooled water allowed very precise determination of Eyring activation enthalpy and entropy from cross relaxation suppressed 2D [(1)H,(1)H]-exchange spectroscopy. This yielded DeltaH = 14 +/- 0.5 kcal.mol(-1) and DeltaS = -4 +/- 1 cal.mol(-1).K(-1), i.e., values close to those previously derived by Wagner and Wüthrich for the temperature range from 4 to 72 degrees C (DeltaH = 16 +/- 1 kcal.mol(-1) and DeltaS = 6 +/- 2 cal.mol(-1).K(-1)). The preservation of the so far uniquely low value for DeltaS indicates that the distribution of internal motional modes associated with the ring flip of Phe 45 is hardly affected by lowering T well below 0 degrees C. Hence, if a globular protein does not cold denature, aromatic flipping rates, and thus likely also the rates of other conformational and/or chemical exchange processes occurring in supercooled water, can be expected to be well estimated from activation parameters obtained at ambient T. This is of keen interest to predict the impact of supercooling for future studies of biological macromolecules, and shows that our approach enables one to conduct NMR-based structural biology at below 0 degrees C in an unperturbed aqueous environment. A search of the BioMagResBank indicated that the overwhelming majority of the Phe and Tyr rings (>95%) are flipping rapidly on the chemical shift time scale at ambient T, while our data for BPTI and activation parameters available for ring-flipping in Iso-2-cytochrome c reveal that in these smaller proteins a total of six out of seventeen rings ( approximately 35%) are "frozen in" at T = -15 degrees C. This suggests that a large fraction of Tyr and Phe rings in globular proteins that are flipping rapidly on the chemical shift time scale at ambient T can be effectively slowed in supercooled water. The present investigation demonstrates that supercooling of protein solutions appears to be an effective means to (i) harvest potential benefits of stalled ring-flipping for refining NMR solution structures, (ii) recruit additional aromatic rings for investigating protein dynamics, and (iii) use multiple slowly flipping rings to probe cold denaturation. The implications for NMR-based structural biology in supercooled water are addressed.

Selective antioxidant enzymes during ischemia/reperfusion in myocardial infarction.

The study of ischemia/reperfusion injury included 25 patients in the acute phase of myocardial infarction (19 perfused, 6 remained non-reperfused as evaluated according to the time course of creatine kinase and CK-MB isoenzyme activity) and a control group (21 blood donors). Plasma level of malondialdehyde was followed as a marker of oxidative stress. Shortly after reperfusion (within 90 min), a transient increase of malondialdehyde concentration was detected. The return to the baseline level was achieved 6 h after the onset of therapy. The activity of a free radical scavenger enzyme, plasma glutathione peroxidase (GPx), reached its maximum 90 min after the onset of treatment and returned to the initial value after 18 h. The specificity of the GPx response was confirmed by comparing with both non-reperfused patients and the control group, where no significant increase was detected. The erythrocyte Cu,Zn-superoxide dismutase (SOD) did not exhibit significant changes during the interval studied in perfused patients, probably due to the stability of erythrocyte metabolism. In non-reperfused patients, a decrease of SOD was found during prolonged hypoxia. These results help to elucidate the mechanisms of fast activation of plasma antioxidant system during the reperfusion after myocardial infarction.

De novo proteins as models of radical enzymes.

Catalytically essential side-chain radicals have been recognized in a growing number of redox enzymes. Here we present a novel approach to study this class of redox cofactors. Our aim is to construct a de novo protein, a radical maquette, that will provide a protein framework in which to investigate how side-chain radicals are generated, controlled, and directed toward catalysis. A tryptophan and a tyrosine radical maquette, denoted alpha(3)W(1) and alpha(3)Y(1), respectively, have been synthesized. alpha(3)W(1) and alpha(3)Y(1) contain 65 residues each and have molecular masses of 7.4 kDa. The proteins differ only in residue 32, which is the position of their single aromatic side chain. Structural characterization reveals that the proteins fold in water solution into thermodynamically stable, alpha-helical conformations with well-defined tertiary structures. The proteins are resistant to pH changes and remain stable through the physiological pH range. The aromatic residues are shown to be located within the protein interior and shielded from the bulk phase, as designed. Differential pulse voltammetry was used to examine the reduction potentials of the aromatic side chains in alpha(3)W(1) and alpha(3)Y(1) and compare them to the potentials of tryptophan and tyrosine when dissolved in water. The tryptophan and tyrosine potentials were raised considerably when moved from a solution environment to a well-ordered protein milieu. We propose that the increase in reduction potential of the aromatic residues buried within the protein, relative to the solution potentials, is due to a lack of an effective protonic contact between the aromatic residues and the bulk solution.

Global topology & stability and local structure & dynamics in a synthetic spin-labeled four-helix bundle protein.

A maleimide nitroxide spin-label (MAL-6) linked to a cysteine in the hydrophobic core and a coproporphyrin I (CP) appended on the N-terminus of a synthetic helix-loop-helix peptide ([alpha2]) have been used to examine the designed self-association of a four-helix bundle ([alpha2]2), focusing on the bundle topology and stability and the rotational dynamics of the spin-label. Gel-permeation chromatography demonstrated that the [alpha2] peptide and the peptide modified with a spin-label ([MAL-6-alpha2]), a coproporphyrin ([CP-alpha2]) and a coproporphyrin plus a spin-label ([CP-MAL-6-alpha2]) self-associate into four helix bundles in solution as designed. Circular dichroism (CD) spectra prove that all these peptides are highly alpha-helical, confirmed for [alpha2]2 by Fourier transform infrared (FTIR) spectroscopic analysis. Electron spin resonance (ESR) spectra of the two attached maleimide spin-labels in [MAL-6-alpha2]2 shows their effective rotational correlation time (tau(c)) is 7.3 +/- 0.5 ns, consistent with that expected for the tumbling of the four helix bundle itself, indicating the labels are immobilized. The ESR spectra were also unaltered by aqueous-phase paramagnetic ions, Ni(II), demonstrating all of the spin-labels are buried within the hydrophobic core. The lack of spin-spin interaction between the buried, immobilized spin-labels indicates they are remote (> 15 A) from each other, indicating an antiparallel topology of the monomers in [MAL-6-alpha2]2. The parent [alpha2]2 and the modified [MAL-6-alpha2]2 and [CP-alpha2]2 peptides are highly stable (deltaG(H2O) approximately 25 kcal/mol) as investigated by guanidine hydrochloride denaturation curves monitored by ESR and CD spectroscopies. Guanidine hydrochloride denaturation leads to a shorter correlation time of the spin-label, tau(c) < 1 ns, approaching that of an unrestricted spin-label in solution. In contrast, trifluoroethanol caused dissociation of [MAL-6-alpha2]2 to yield two [MAL-6-alpha2] monomers with retention of secondary structure and changed the tau(c) to 2.5 +/- 0.5 ns, indicating that a significant degree of motional restriction is imposed on the spin-label by the secondary structure. The coproporphyrin probes covalently attached to the N-termini of [CP-alpha2]2 and [CP-MAL-6-alpha2]2 provided evidence that the helical monomers of both were in a parallel orientation, in contrast to the antiparallel orientation determined for [MAL-6-alpha2]2. Consequently, the ESR spectra of [MAL-6-alpha2]2 and [CP-MAL-6-alpha2]2 reveal major structural differences in the local vicinity of the spin-labels due to the topological difference between these two bundles. The ESR spectra of [CP-MAL-6-alpha2]2 contains two distinct nitroxide populations, indicating that one spin-label remains buried in the hydrophobic core and the other is excluded to solvent in this parallel topology. Alleviation of the steric interactions causing one spin-label in [CP-MAL-6-alpha2]2 to be solvent-exposed by addition of [CP-alpha2]2 results in formation of the heterodimeric [CP-alpha2]/[CP-MAL-6-alpha2], as evidenced by insertion of all the spin-labels into hydrophobic cores. The changes in global topology and local structure as evidenced by this pair of spectral probes have relatively minor effects on the course of guanidine denaturation of these bundles.

Structural coupling of the inhibitory regions flanking the ETS domain of murine Ets-1.

Several members of the ets gene family of transcription factors show negative regulation of DNA binding by intramolecular interactions. A structural mechanism for this auto-inhibition is investigated using a 161-residue N-terminal deletion mutant of murine Ets-1, Ets-1 delta N280. This protein shows a similar reduced affinity for DNA as native Ets-1 because it contains the ETS domain in context of the flanking amino- and carboxy-terminal regions that together mediate repression of DNA binding. The secondary structure of Ets-1 delta N280 was determined using NMR chemical shift, NOE, J coupling, and amide hydrogen exchange information. In addition to the winged helix-turn-helix ETS domain, Ets-1 delta N280 contains two alpha-helices in the amino-terminal inhibitory region and one alpha-helix in the carboxy-terminal inhibitory region. Chemical shift comparisons were made between this protein and an activated form of Ets-1 lacking the amino-terminal inhibitory region. The spectral differences demonstrate that the amino- and carboxy-terminal inhibitory sequences are structurally coupled to one another, thus explaining the observation that both regions are required for the repression of DNA binding. Furthermore, these data show that the inhibitory sequences also interact directly with the first helix of the intervening ETS domain, thereby providing a pathway for the repression of DNA binding. These results lead to a model of an inhibitory module in Ets-1 composed of both the amino- and carboxy-terminal regions interfaced with the ETS domain. This establishes the structural framework for understanding the intramolecular inhibition of Ets-1 DNA binding.

Modulation of transcription factor Ets-1 DNA binding: DNA-induced unfolding of an alpha helix.

Conformational changes, including local protein folding, play important roles in protein-DNA interactions. Here, studies of the transcription factor Ets-1 provided evidence that local protein unfolding also can accompany DNA binding. Circular dichroism and partial proteolysis showed that the secondary structure of the Ets-1 DNA-binding domain is unchanged in the presence of DNA. In contrast, DNA allosterically induced the unfolding of an alpha helix that lies within a flanking region involved in the negative regulation of DNA binding. These findings suggest a structural basis for the intramolecular inhibition of DNA binding and a mechanism for the cooperative partnerships that are common features of many eukaryotic transcription factors.

Mapping receptor binding sites in interleukin (IL)-1 receptor antagonist and IL-1 beta by site-directed mutagenesis. Identification of a single site in IL-1ra and two sites in IL-1 beta.

Interleukin-1 receptor antagonist (IL-1ra), an IL-1 family member, binds with high affinity to the type I IL-1 receptor (IL-1RI), blocking IL-1 binding but not inducing an IL-1-like response. Extensive site-directed mutagenesis has been used to identify residues in IL-1ra and IL-1 beta involved in binding to IL-1RI. These analyses have revealed the presence of two discrete receptor binding sites on IL-1 beta. Only one of these sites is present on IL-1ra, consisting of residues Trp-16, Gln-20, Tyr-34, Gln-36, and Tyr-147. Interestingly, the absent second site is at the location of the major structural difference between IL-1ra and IL-1 beta, which are otherwise structurally similar. The two receptor binding sites on IL-1 beta are also present on IL-1 alpha. Thus, it appears that the two IL-1 agonist molecules have two sites for IL-1RI binding, and the homologous antagonist molecule, IL-1ra, has only one. Based on these observations, a hypothesis is presented to account for the difference in activity between the agonist and antagonist proteins. It is proposed that the presence of the two receptor binding sites may be necessary for agonist activity.

Structure and dynamics of the neutrophil defensins NP-2, NP-5, and HNP-1: NMR studies of amide hydrogen exchange kinetics.

The exchange kinetics for the slowly exchanging amide hydrogens in three defensins, rabbit NP-2, rabbit NP-5, and human HNP-1, have been measured over a range of pH at 25 degrees C using 1D and 2D NMR methods. These NHs have exchange rates 10(2) to 10(5) times slower than rates from unstructured model peptides. The observed distribution of exchange rates under these conditions can be rationalized by intramolecular hydrogen bonding of the individual NHs, solvent accessibility of the NHs, and local fluctuations in structure. The temperature dependencies of NH chemical shifts (NH temperature coefficients) were measured for the defensins and these values are consistent with the defensin structure. A comparison is made between NH exchange kinetics, NH solvent accessibility, and NH temperature coefficients of the defensins and other globular proteins. Titration of the histidine side chain in NP-2 was examined and the results are mapped to the three-dimensional structure.

Solution structure of the calcium channel antagonist omega-conotoxin GVIA.

The three-dimensional solution structure is reported for omega-conotoxin GVIA, which is a potent inhibitor of presynaptic calcium channels in vertebrate neuromuscular junctions. Structures were generated by a hybrid distance geometry and restrained molecular dynamics approach using interproton distance, torsion angle, and hydrogen-bonding constraints derived from 1H NMR data. Conformations of GVIA with low constraint violations converged to a common peptide fold. The secondary structure in the peptide is an antiparallel triple-stranded beta-sheet containing a beta-hairpin and three tight turns. The NMR data are consistent with the region of the peptide from residues S9 to C16 being more dynamic than the rest of the peptide. The peptide has an amphiphilic structure with a positively charged hydrophilic side and an opposite side that contains a small hydrophobic region. Residues that are thought to be important in binding and function are located on the hydrophilic face of the peptide.

NMR studies of defensin antimicrobial peptides. 2. Three-dimensional structures of rabbit NP-2 and human HNP-1.

The solution structure of two homologous naturally occurring antimicrobial peptides, rabbit defensin NP-2 and human defensin HNP-1, have been determined by two-dimensional nuclear magnetic resonance spectroscopy, distance geometry, and restrained molecular dynamics calculations. The structure of these defensins consists of an antiparallel beta-sheet in a hairpin conformation, a short region of triple-stranded beta-sheet, several tight turns, and a loop region that has a well-defined local structure but with a global orientation that is not well-defined with respect to the rest of the molecule. The solution structures of these two peptides are compared with the solution and crystal structures of two other homologous defensins. The structures for the defensins are also compared with known structures of other naturally occurring antimicrobial peptides.

[Several physiologic aspects of the reactions of foals in the first three days after weaning]. / Einige physiologische Gesichtspunkte der Reaktion von Fohlen in den ersten drei Tagen nach dem Absetzen.

Changes have been observed in nine foals, six months of age, after weaning for the time of three days, namely in the intake of drinking water, hematocrit, sedimentation of erythrocytes and three minerals Na+, K+ and Cl-. Foals respond to weaning immediately, lowering significantly their consumption of drinking water and increasing high significance of Na+ and Cl-. Changes of level K+, hematocrit and sedimentation don't show any significant variations. The results reached are to be considered as an introductory information for further inquiry on the more extensive methodical basis.

Membrane-associated protein kinase activities in seminal plasma from vasectomized men.

Differential centrifugation was used to prepare heavy and light membrane fractions from the seminal plasma of vasectomized men. The two membrane fractions combined contained half of the phosvitin and histone kinase activities but only 7% of the total protein content in vasectomy semen. These two kinase activities as well as phosphorylation of endogenous membrane proteins were optimally stimulated by Mg2+; Mn2+ could effectively substitute for Mg2+ only in endogenous phosphorylation reactions. Neither the phosvitin nor histone kinase responded to cAMP or cGMP, but the histone kinase was strongly inhibited by the heat-stable cAMP-dependent protein kinase inhibitor. The phosvitin kinase was not affected by this inhibitor. The phosphorylation of endogenous proteins in the heavy membrane fraction was not affected by the protein kinase inhibitor but protein phosphorylation in the light membrane fraction was partly (45%) inhibited. The differential effects of increased ionic strength, sulphydryl protecting agents, and the protein kinase inhibitor on protein kinase activity towards lysine-rich histones, phosvitin and endogenous proteins, as well as differential extractability and binding to an anion exchange column of histone kinase and phosvitin kinase activities, indicate that more than one kinase activity is present in these membrane subfractions. Electron microscopic examination showed that there are several kinds of membrane-limited components in vasectomy seminal fluid that vary in size, density, and ultrastructure. The association of type(s) of protein kinase to individual membrane components remains to be established.