Application of tritium high resolution NMR spectroscopy to analysis of tritium-labelled amino acids and peptides

Summary A method has been developed for the qualitative and quantitative analysis of complex isotopic mixtures of tritium-labelled amino acids and peptides by using high resolution³H NMR spectroscopy at 266.8 MHz. Determined were tritium distribution in alanine, glycine, tryptophan and 4-hydroxyproline amino acids, as well as in glycine and valine residues of peptides. Approaches have been worked out for the determination of spin coupling constants and isotope chemical shifts for the strongly coupled nonequivalent atoms of the methylene groups.     

Amino acid exchange between plasma and erythrocytes in vivo in humans

To study amino acid exchange between plasma and erythrocytes in vivo, 4-h primed, continuous intravenous infusions of L-[1-13C]leucine, [15N]glycine, and L-[15N]alanine were administered to five healthy young men in the postabsorptive state. Stable isotope enrichments and amino acid levels were determined by gas chromatography-mass spectrometry in both plasma and whole blood and estimated (using hematocrit) in erythrocytes. A high concentration gradient across the erythrocyte membrane was consistently found for glycine (552 +/- 268 microM in erythrocytes vs. 155 +/- 35 microM in plasma), but not for leucine or alanine. A steady-state isotopic enrichment was observed in whole blood as well as plasma for each amino acid in every subject. Steady-state [13C]leucine enrichment in erythrocytes did not differ from plasma enrichment at steady state, the ratio of erythrocyte to plasma enrichment being 1.03 +/- 0.20 (95% confidence limits = 0.78-1.28); in contrast, this ratio reached only 0.23 +/- 0.04 and 0.59 +/- 0.09 (confidence limits 0.18-0.28 and 0.48-0.70) for [15N]glycine and [15N]alanine at steady state, respectively. These results suggest that most of erythrocyte leucine is exchangeable with plasma, whereas only a fraction of erythrocyte glycine and alanine is involved in exchange with plasma in vivo.     

Purification by affinity chromatography of the glycine receptor of rat spinal cord

The glycine receptor of rat spinal cord was solubilized with the nonionic detergent Triton X-100 and subsequently purified by affinity chromatography on aminostrychnine-agarose and wheat germ agglutinin-Sepharose. An overall purification of 1950-fold was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol revealed three glycine receptor-associated polypeptides of Mr = 48,000, 58,000, and 93,000. [3H]Strychnine was incorporated irreversibly into the Mr = 48,000 polypeptide upon UV-illumination. The dissociation constant (KD) of [3H]strychnine binding to the purified glycine receptor was 9.3 +/- 0.6 nM. The glycine receptor agonists glycine, beta-alanine, and taurine inhibited the binding of [3H]strychnine to the purified receptor. Gel filtration and sedimentation in sucrose/H2O and sucrose/D2O gradients gave a Stokes radius of 7.7 nm, a partial specific volume of 0.780 +/- 0.005 ml/g and a sedimentation coefficient s20,w of 8.2 +/- 0.2 S for the purified glycine receptor. From these data, a molecular weight of 246,000 +/- 6,000 was calculated for the glycine receptor protein.     

Computer analysis of double-labeled two-dimensional electrophoresis gels

A computerized process for the automatic analysis of double-label autoradiography after two-dimensional polyacrylamide gel electrophoresis has been developed. Matching fluorographs and autoradiographs produced from gels containing 3H- and 14C-labeled proteins are digitized by a rotating drum densitometer and analyzed by the Man-computer Interactive Data Analysis System III. This system locates corresponding protein spots in the films with edge-detection algorithms, converts spot density readings to isotopic disintegrations by reference to standard curves, and computes a 3H:14C ratio for each spot in the gels. On the average, calculated ratios are accurate to approximately 9% for test strips of polyacrylamide gel containing uniform mixtures of 3H and 14C. Values obtained for two-dimensional gels containing n protein spots with a known 3H:14C ratio of 8.6 +/- 0.1 are as follows: 8.1 +/- 1.4 (n = 268), 8.8 +/- 2.1 (n = 278), 9.1 +/- 1.7 (n = 245), and 8.8 +/- 2.2 (n = 223). The computer process greatly reduces the time required to precisely compare two complex protein mixtures and has sufficient precision to detect a doubling in the biosynthesis of any individual protein.     

A 13C NMR study of [5,8-13C2]spermidine binding to tRNA and to Escherichia coli macromolecules

[5,8-13C2]Spermidine was prepared by synthesis, and its binding to macromolecular structures of Escherichia coli was studied. When added to E. coli cells, the two signals of [13C]spermidine (C-5, 47.8 ppm, and C-8, 39.6 ppm; JC-C = 5.8 Hz) were strongly broadened due to binding to macromolecules. When [13C]spermidine was added to E. coli tRNA, the C-5 resonance broadened to v1/2 = 4.7 Hz, whereas the C-8 resonance broadened to v1/2 = 2.7 Hz. tRNA-bound [13C]spermidine could be chased by [12C]spermidine or spermine, but not by putrescine or cadaverine. By using mixtures of [5-13C]- and [8-13C]spermidines (where 13C-13C coupling was avoided), it was possible to estimate a dissociation constant (Kd) of 3 x 10(-3) M using the C-5 v1/2obs values and a Kd of 2.10(-3) M using the C-8 v1/2obs values. The number of spermidine-binding sites (n) could also be estimated by fitting the bound spermidine molar fraction versus tRNA concentration. Values of n = 12 +/- 2 and 14 +/- 3 were obtained for C-5 and C-8, respectively. Measurements of line narrowing at increasing Mg2+ concentrations indicated that approximately 11 spermidines (of the 12-14 bound ones) could be displaced by the former, whereas 3 spermidines remain strongly bound to the tRNA backbone. Measurements of free and bound T1 allowed the determination of a correlation time of 10(-10)s for tRNA-bound spermidine.     

Frequency-domain characteristics and filtering of blood flow following the onset of exercise: implications for kinetics analysis.

We examined the validity and usefulness of a low-pass filter (LPFILTER) to reduce point-to-point variability and enhance parameter estimation of the kinetics of blood flow (BF). Computer simulations were used to determine the power spectrum of simulated responses. Moreover, we studied the leg BF response to a single transition in four subjects during supine knee-extension exercise using three methods of data processing [beat-by-beat, average of 3 cardiac cycles (AVG3 BEATS), and LPFILTER]. The power spectrum of BF containing the kinetics information (0.05; n=4). However, LPFILTER (cutoff=0.2 Hz) resulted in a significantly lower standard error of the estimate for all parameters (P<0.05). The means+/-SD for the standard error of the estimate for Beat-by-Beat, AVG3 BEATS, and LPFILTER were, respectively, time constant-phase 1=5.0+/-1.1 s, 4.5+/-2.1 s, and 0.3+/-0.2 s; time delay-phase 2=17.8+/-7.9 s, 12.8+/-7.5 s, and 1.4+/-1.4 s; time constant-phase 2=15.8+/-4.6 s, 9.9+/-2.9 s, and 1.1+/-0.5 s. In conclusion, LPFILTER appeared to be a valid procedure providing a high signal-to-noise ratio and data density and thus LPFILTER resulted in the smallest confidence interval for parameter estimates of BF kinetics.     

An inquiry into the selective protection of glycine during the radiolysis of glycine-alanine mixtures in aqueous solutions and its implications to the preservation of optically active amino acids in the early earth

Akaboshi et al. (1990) has found an unexpected protection of the achiral amino acid, glycine, towards ionizing radiation at the expense of the selective destruction of the chiral amino acids, alanine and aspartic acid. The present work examines the mechanism of this protection for the case of alanine. We have developed a computer model for the radiolysis of glycine, alanine and glycine-alanine mixtures in aqueous solution. It is established that this protection is due in part to the reaction of the α-radical of glycine with alanine to regenerate a more stable α-radical, according to the following reaction, $$ \cdot CH(NH_3^ + )CO_2^ - + CH_3 CH(NH_3^ + )CO_2^ - \to CH_2 (NH_3^ + )CO_2^ - + CH_3 \dot C(NH_3^ + )CO_2^ -$$ The rate constant of this reaction was estimated to be ≤10⁴M^-1s^-1. The implications for this selective protection of glycine are considered for a hypothetical case in which there would be an enrichment of about 10% ofL-alanine in the primitive ocean and taking the glycine/alanine ratios obtained in CH₄-and CO₂- dominated atmospheres using electric discharge experiments. It is predicted that alanine would be rapidly destroyed and radioracemized in spite of the fact that the concentration of alanine is equal or significantly lower than that of glycine. Assuming that chiral amino acids were a prerequisite for the origin of life, it can be deduced that life could have appeared in a relatively short period of time unless there was a constant supply of optical amino acids from extraterrestrial sources.     

Hypothalamic mapping of orexigenic action and Fos-like immunoreactivity following relaxin-3 administration in male Wistar rats

The insulin superfamily, characterized by common disulphide bonds, includes not only insulin but also insulin-like peptides such as relaxin-1 and relaxin-3. The actions of relaxin-3 are largely unknown, but recent work suggests a role in regulation of food intake. Relaxin-3 mRNA is highly expressed in the nucleus incertus, which has extensive projections to the hypothalamus, and relaxin immunoreactivity is present in several hypothalamic nuclei. In the rat, relaxin-3 binds and activates both relaxin family peptide receptor 1, which also binds relaxin-1, and a previously orphaned G protein-coupled receptor, RXFP3. These receptors are extensively expressed in the hypothalamus. The aims of these studies were twofold: 1) map the hypothalamic site(s) of the orexigenic action of relaxin-3 and 2) examine the site(s) of neuronal activation following central relaxin-3 administration. After microinjection into hypothalamic sites, human relaxin-3 (H3; 180 pmol) significantly stimulated 0- to 1-h food intake in the supraoptic nucleus (SON), arcuate nucleus (ARC), and the anterior preoptic area (APOA) [SON 0.4+/-0.2 (vehicle) vs. 2.9+/-0.5 g (H3), P<0.001; ARC 0.7+/-0.3 (vehicle) vs. 2.7+/-0.2 g (H3), P<0.05; and APOA 0.8+/-0.1 (vehicle) vs. 2.2+/-0.2 g (H3), P<0.05]. Cumulative food intake was significantly increased相似文献   

Modulation of airway caliber by deep inhalation in children.

To elucidate whether deep inhalation (DI) modulates changes in airway caliber in childhood, we measured the effect of DI on respiratory impedance before and after inhaled methacholine or salbutamol in 4- to 7-yr-old children (n = 15) suffering from recurrent wheezing. In all children, the real part of impedance between 12 and 16 Hz (Re[Z](12-16)) increased after methacholine from 5.6 +/- 1.2 to 8.2 +/- 1.6 cmH(2)O. l(-1). s (P < 0.001) and resonance frequency from 18 +/- 3 to 25 +/- 5 Hz (P < 0.001). These changes were partially reversed by DI: Re[Z](12-16) decreased to 7.2 +/- 1.2 cmH(2)O. l(-1). s (P < 0.01) and resonance frequency to 19 +/- 5 Hz (P < 0.001). In nine children, on a separate occasion, Re[Z](12-16) decreased after salbutamol from 8.3 +/- 1.9 to 5.1 +/- 0.9 cmH(2)O. l(-1). s (P < 0.001) and resonance frequency from 21 +/- 6 to 15 +/- 3 Hz (P < 0.05). The decrease of Re[Z](12-16) was partially reversed by DI (to 6.2 +/- 1.4 cmH(2)O. l(-1). s, P < 0. 01), but resonance frequency did not change significantly (P = 0.75). We conclude that in 4- to 7-yr-old children pharmacologically induced changes in airway caliber are modulated by DI. These findings suggest that airway-to-parenchyma interdependence is operative in this age range.     

Inhibition of hepatic proteolysis by insulin. Role of hormone-induced alterations of the cellular K+ balance

1. Proteolysis was measured as [3H]leucine release from isolated perfused livers from rats, which had been labeled in vivo by an intraperitoneal injection of [3H]leucine about 16 h prior to the perfusion experiment. In livers from fed rats, insulin (35 nM) inhibited [3H]leucine release by 24.5 +/- 1.3% (n = 15) and led to an amiloride-sensitive, bumetanide-sensitive and furosemide-sensitive net K+ uptake of 5.53 +/- 0.31 mumol.g-1 (n = 15). Both the insulin effects on net K+ uptake and on [3H]leucine release were diminished by about 65% or 55% in presence of furosemide (0.1 mM) or bumetanide (5 microM), respectively. The insulin-induced net K+ uptake was virtually abolished in the presence of amiloride (1 mM) plus furosemide (0.1 mM). 2. In perfused livers from 24-h-starved rats, both the insulin-stimulated net K+ uptake and the insulin-induced inhibition of [3H]leucine release were about 80% lower than observed in experiments with livers from fed rats. The insulin effects on K+ balance and [3H]leucine release were not significantly influenced in the presence of glycine (2 mM), although glycine itself inhibited [3H]leucine release by 30.3 +/- 0.3% (n = 4) and 13.8 +/- 1.2% (n = 5) in livers from starved and fed rats, respectively. When livers from fed rats were preswollen by hypoosmotic perfusion (225 mOsmol.l-1), both the insulin-induced net K+ uptake and the inhibition of [3H]leucine release were diminished by 50-60%. 3. During inhibition of [3H]leucine release by insulin, further addition of glucagon (100 nM) led to a marked net K+ release from the liver (3.82 +/- 0.24 mumol.g-1), which was accompanied by stimulation of [3H]leucine release by 16.4 +/- 4.6% (n = 4). 4. Ba2+ (1 mM) infusion led to a net K+ uptake by the liver of 3.2 +/- 0.2 mumol.g-1 (n = 4) and simultaneously inhibited [3H]leucine release by 12.4 +/- 1.7% (n = 4). 5. There was a close relationship between the Ba2+ or insulin-induced net K+ uptake and the degree of inhibition of [3H]leucine release, even when the K+ response to insulin was modulated by bumetanide, furosemide, glucagon, hypotonic or glycine-induced cell swelling or the nutritional state. 6. The data suggest that the insulin-induced net K+ uptake involves activation of both NaCl/KCl cotransport and Na+/H+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanical impedances of lungs and chest wall in the cat.

In nine anesthetized and paralyzed cats, the mechanical impedances of the total respiratory system (Zrs) and the lungs (ZL) were measured with small-volume pseudorandom forced oscillations between 0.2 and 20 Hz. ZL was measured after thoracotomy, and chest wall impedance (Zw) was calculated as Zw = Zrs-ZL. All impedances were determined by using input airflow [input impedance (Zi)] and output flow measured with a body box [transfer impedance (Zt)]. The differences between Zi and Zt were small for Zrs and negligible for ZL. At 0.2 Hz, the real and imaginary parts of ZL amounted to 33 +/- 4 and 35 +/- 3% (SD), respectively, of Zrs. Up to 8 Hz, all impedances were consistent with a model containing a frequency-independent resistance and inertance and a constant-phase tissue part (G-jH)/omega alpha, where G and H are coefficients for damping and elastance, respectively, omega is angular frequency, and alpha determines the frequency dependence of the real and imaginary parts. G/H was higher for Zw than for ZL (0.29 +/- 0.05 vs. 0.22 +/- 0.04, P less than 0.01). In four cats, the amplitude dependence of impedances was studied: between oscillation volumes of 0.8 and 3 ml, GL, HL, Gw, and Hw decreased on average by 3, 9, 26, and 29%, respectively, whereas the change in G/H was small for both ZL (7%) and Zw (-4%). The values of H were two to three times higher than the quasistatic elastances estimated with greater volume changes (greater than 20 ml).     

Detect of Escherichia coli O157:H7 in ground beef samples using piezoelectric excited millimeter-sized cantilever (PEMC) sensors

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors consisting of a piezoelectric and a borosilicate glass layer with a sensing area of 4 mm2 were fabricated. An antibody specific to Escherichia coli (anti-E. coli) O157:H7 was immobilized on PEMC sensors, and exposed to samples containing E. coli O157:H7 (EC) prepared in various matrices: (1) broth, broth plus raw ground beef, and broth plus sterile ground beef without inoculation of E. coli O157:H7 served as controls, (2) 100 mL of broth inoculated with 25 EC cells, (3) 100 mL of broth containing 25 g of raw ground beef and (4) 100 mL of broth with 25 g of sterile ground beef inoculated with 25 EC cells. The total resonant frequency change obtained for the broth plus EC samples were 16+/-2 Hz (n=2), 30 Hz (n=1), and 54+/-2 Hz (n=2) corresponding to 2, 4, and 6h growth at 37 degrees C, respectively. The response to the broth plus 25 g of sterile ground beef plus EC cells were 21+/-2 Hz (n=2), 37 Hz (n=1), and 70+/-2 Hz (n=2) corresponding to 2, 4, and 6 h, respectively. In all cases, the three different control samples yielded a frequency change of 0+/-2 Hz (n=6). The E. coli O157:H7 concentration in each broth and beef samples was determined by both plating and by pathogen modeling program. The results indicate that the PEMC sensor detects E. coli O157:H7 reliably at 50-100 cells/mL with a 3 mL sample.     

Hypertension and impaired glycine handling in mice lacking the orphan transporter XT2

A family of orphan transporters has been discovered that are structurally related to the Na(+)-Cl(-)-dependent neurotransmitter transporters, including the dopamine transporter. One member of this family, the mouse XT2 gene, is predominantly expressed in the kidney and has 95% homology to rat ROSIT (renal osmotic stress-induced Na(+)-Cl(-) organic solute cotransporter). To study the physiological functions of this transporter, we generated XT2-knockout mice by gene targeting. XT2(-/-) mice develop and survive normally with no apparent abnormalities. To attempt to identify potential substrates for XT2, we screened urine from XT2-knockout mice by high-pressure liquid chromatography and mass spectrometry and found significantly elevated concentrations of glycine. To study glycine handling, XT2(+/+) and XT2(-/-) mice were injected with radiolabeled glycine, and urine samples were collected to monitor glycine excretion. After 2 h, XT2(-/-) mice were found to excrete almost twice as much glycine as the XT2(+/+) controls (P = 0.03). To determine whether the absence of the XT2 transporter affected sodium and fluid homeostasis, we measured systolic blood pressure by computerized tail-cuff manometry. Systolic blood pressure was significantly higher in XT2(-/-) mice (127 +/- 3 mmHg) than in wild-type controls (114 +/- 2 mmHg; P < 0.001). This difference in systolic blood pressure was maintained on high and low salt feeding. To examine whether the alteration in blood pressure and the defect in glycine handling were related, we measured systolic blood pressure in the XT2(-/-) mice during dietary glycine supplementation. Glycine loading caused systolic blood pressure to fall in the XT2(-/-) mice from 127 +/- 3 to 115 +/- 3 mmHg (P < 0.001), a level virtually identical to that of the wild-type controls. These data suggest that the XT2 orphan transporter is involved in glycine reabsorption and that the absence of this transporter is sufficient to cause hypertension.     

Effects of glycine and alanine on short-term storage and cryopreservation of striped bass (Morone saxatilis) spermatozoa

Three experiments were designed to examine the effects of the amino acids glycine and alanine on short-term storage and cryopreservation of striped bass spermatozoa. In the first experiment, the effect of glycine on post-equilibration motility was evaluated. In the presence of 2.5 or 5.0% Me(2)SO, glycine treatments (25, 50, and 75 mM) yielded higher (P<0.05) post-equilibration motility at all equilibration times examined compared to the control. There was no difference (P>0.05) among these three glycine treatments. In the second experiment, glycine and alanine at concentrations of 25, 50, 75, or 100mM were evaluated for post-thaw motility in the presence of 2.5 or 5% Me(2)SO. When compared to the control, both the glycine and alanine treatments showed positive effects on post-thaw motility at all concentrations tested. The highest (P<0.05) post-thaw motility was achieved with 50mM glycine or 75 mM alanine using 5% Me(2)SO. No interaction (P>0.05) between Me(2)SO and glycine or alanine was observed, indicating that the effect of glycine or alanine was independent of the concentrations of Me(2)SO. In the third experiment, glycine was evaluated for sperm motility, after short-term refrigerated storage and after cryopreservation of the same refrigerated semen. Sperm motility decreased after 24h of refrigerated storage in 50mM glycine treatment and the control, when compared to fresh sperm motility. However, 50mM glycine treatment yielded higher (P<0.01) sperm motility after both 24 and 48 h of storage as well as higher (P<0.01) post-thaw motility when compared to the control. An average of 30+/-2.9% and 16+/-2.4% post-thaw motility was achieved with the 50mM glycine treatment after 24 and 48 h of refrigerated semen, respectively.     

Effect of exercise-induced arterial hypoxemia on quadriceps muscle fatigue in healthy humans

The effect of exercise-induced arterial hypoxemia (EIAH) on quadriceps muscle fatigue was assessed in 11 male endurance-trained subjects [peak O2 uptake (VO2 peak) = 56.4 +/- 2.8 ml x kg(-1) x min(-1); mean +/- SE]. Subjects exercised on a cycle ergometer at >or=90% VO2 peak) to exhaustion (13.2 +/- 0.8 min), during which time arterial O2 saturation (Sa(O2)) fell from 97.7 +/- 0.1% at rest to 91.9 +/- 0.9% (range 84-94%) at end exercise, primarily because of changes in blood pH (7.183 +/- 0.017) and body temperature (38.9 +/- 0.2 degrees C). On a separate occasion, subjects repeated the exercise, for the same duration and at the same power output as before, but breathed gas mixtures [inspired O2 fraction (Fi(O2)) = 0.25-0.31] that prevented EIAH (Sa(O2) = 97-99%). Quadriceps muscle fatigue was assessed via supramaximal paired magnetic stimuli of the femoral nerve (1-100 Hz). Immediately after exercise at Fi(O2) 0.21, the mean force response across 1-100 Hz decreased 33 +/- 5% compared with only 15 +/- 5% when EIAH was prevented (P < 0.05). In a subgroup of four less fit subjects, who showed minimal EIAH at Fi(O2) 0.21 (Sa(O2) = 95.3 +/- 0.7%), the decrease in evoked force was exacerbated by 35% (P < 0.05) in response to further desaturation induced via Fi(O2) 0.17 (Sa(O2) = 87.8 +/- 0.5%) for the same duration and intensity of exercise. We conclude that the arterial O2 desaturation that occurs in fit subjects during high-intensity exercise in normoxia (-6 +/- 1% DeltaSa(O2) from rest) contributes significantly toward quadriceps muscle fatigue via a peripheral mechanism.     

Analysis of highly tritium-labeled alanine using 3H- and 1H-NMR

A method has been developed for the analysis of deuterium oxide solutions of tritiated alanine eight-component isotopic mixtures by using high resolution 3H and 1H NMR spectra at frequencies 266.8 and 500.13 MHc, respectively. Approaches have been worked out for the determination of qualitative composition of the mixtures and spectral parameters.     

Reliability in estimates of body composition of birds: oxygen-18 versus deuterium dilution

Body composition in birds was evaluated indirectly by 18O and 2H dilution. Body composition was determined by whole-body chemical analysis of eight adult roosters (Gallus gallus). In vivo measurements of total body water (TBW) were carried out using doubly labeled water (2H2 18O). Estimated dilution spaces using both the plateau and intercept approaches were compared with the results obtained by carcass lyophilization. Both 18O and 2H slightly overestimated TBW compared with the results obtained by lyophilization, by 2.2%+/-1.9% and 5.7%+/-0.2%, respectively; both differences were statistically significant (P<0.01). The difference between these isotope estimations was significant (P<0.001). However, isotope dilution spaces and TBW were highly correlated. There was a strong inverse relationship between total body fat and TBW percentages (r2=0.98, P<0.0001). The relation between TBW and body protein was significant. Water content in lean body mass (72.8%) obtained in our study was very close to that reported in mammals, demonstrating no fundamental difference in tissue water content between birds and mammals. Estimated body fat and protein values from isotopic dilution did not significantly differ from values obtained by direct chemical analysis (P>0.05), except for body fat in the Pace and Rathbun approach (Table 3). Although estimation of TBW and body composition by isotope dilution is time consuming and expensive, deuterium offers a reliable and low-cost alternative compared with 18O. The advantage of in vivo estimation of TBW with isotopic dilution in combination with the regression approach is that it permits repeated measurements of body composition on the same birds under laboratory and free-living conditions.     

Mixed Chelates of Ca(II)-Pyridine-2,6-Dicarboxylate with Some Amino Acids Related to Bacterial Spores

The high resistance of bacterial spores to heat has been repeatedly postulated to be due to stabilization of spore biopolymers by metal chelate compounds. Binding of calcium dipicolinic acid (Ca(II)-DPA) with spore proteins and amino acids has been discussed in the literature, but equilibrium data are generally lacking. By means of potentiometric pH titrations at 25 degrees C and an ionic strength of 1.0 (KNO(3)), the formation of Ca(II)-DPA (1:1 and 1:2) chelates and the interactions of Ca(II)-DPA chelate with a mole of each of three typical amino acids viz., cysteine, alanine, and glycine has been investigated. Analysis of the potentiometric data indicates that calcium and DPA forms 1:1 and 1:2 chelates with log K(ML1) = 4.39 +/- 0.01 and log K(ML2) = 2.25 +/- 0.01. In the presence of an equimolar amount of each of the amino acids under consideration, the Ca(II)-DPA chelate forms mixed ligand (ternary) chelate yielding the following stepwise stability constants: log K(1) = 4.17 +/- 0.01, log K(2) = 0.78 +/- 0.01 for cysteine, log K(1) = 4.06 +/- 0.01, log K(2) = 0.65 +/- 0.01 for alanine, and log K(1) = 4.30 +/- 0.02, log K(2) = 0.11 +/- 0.01 for glycine. Methods for calculating the stability constants of the mixed ligand system have been developed. On the basis of the potentiometric equilibrium data, possible structures for the various calcium chelate species are discussed. The data suggest that the differences in heat resistance of various strains of bacterial spores may conceivably be related to the differences in composition and stability of coordination complexes in the spore.     

Skin blood flowmotion and microvascular reactivity investigation in hypercholesterolemic patients without clinically manifest arterial diseases

Fourier spectral analysis of forearm skin laser Doppler flowmetry (LDF) signal was performed in fifteen hypercholesterolemic patients (HP), without clinically manifest arterial diseases, and in fifteen age-matched healthy control subjects (CS), in order to investigate skin blood flowmotion (SBF). The LDF frequency intervals studied were: 0.01-1.6 Hz total spectrum, as well as 0.01-0.02 Hz (endothelial), 0.02-0.06 Hz (sympathetic), 0.06-0.2 Hz (myogenic), 0.2-0.6 Hz (respiratory) and 0.6-1.6 Hz (cardiac). Skin microvascular reactivity (MVR) to acetylcholine (ACh) and to sodium nitroprusside (SNP) iontophoresis was also investigated. HP showed a lower post-ACh increase in power spectral density (PSD) of the 0.01-0.02 Hz SBF subinterval compared to CS (1.80+/-1.73 PU(2)/Hz vs 3.59+/-1.78 PU(2)/Hz, respectively; p<0.005), while they did not differ in MVR from CS. In eleven HP the 0.01-0.02 Hz SBF subinterval showed a higher post-ACh PSD increase near to the statistical significance after 10 weeks of rosuvastatin therapy (10 mg/day) compared to pretreatment test (3.04+/-2.95 PU(2)/Hz vs 1.91+/-1.94 PU(2)/Hz; p=0.07). The blunted post-ACh increase in PSD of the 0.01-0.02 Hz SBF subinterval in HP suggests a skin endothelial dysfunction in these patients. This SBF abnormality showed a tendency to improve after rosuvastatin therapy in eleven treated patients.     

Approved quarantine treatment for Hessian fly (Diptera: Cecidomyiidae) in large-size hay bales and Hessian fly and cereal leaf beetle (Coleoptera: Chrysomelidae) control by bale compression

A quarantine treatment using bale compression (32 kg/cm2 pressure) and phosphine fumigation (61 g/28.3 m3 aluminum phosphide for 7 d at 20 degrees C) was approved to control Hessian fly, Mayetiola destructor (Say), in large-size, polypropylene fabric-wrapped bales exported from the western states to Japan. No Hessian fly puparia (45,366) survived to the adult stage in infested wheat, Triticum aestivum L., seedlings exposed to the treatment in a large-scale commercial test. Daily temperatures (mean +/- SEM) inside and among bales in three test freight containers were 17.8 +/- 0.2 front top, 17.0 +/- 0.2 front bottom, 17.3 +/- 0.2 middle bale, 15.7 +/- 0.3 middle air, 18.5 +/- 0.1 back top, and 18.1 +/- 0.1 degrees C back bottom, allowing the fumigation temperature to be established at > or = 20 degrees C. Mean fumigant concentrations ranged from 208 to 340 ppm during the first 3 d and ranged from 328 to 461 ppm after 7 d of fumigation. Copper plate corrosion values inside the doors, and in the middle of the large-size bales in all locations indicated moderate exposure to hydrogen phosphide (PH3). PH3 residues were below the U.S. Environmental Protection Agency tolerance of 0.1 ppm in animal feeds. The research was approved by Japan and U.S. regulatory agencies, and regulations were implemented on 20 May 2005. Compression in large-size bale compressors resulted in 3-3.6 and 0% survival of Hessian fly puparia and cereal leaf beetle, Oulema melanopus (L.), respectively. Bale compression can be used as a single treatment for cereal leaf beetle and as a component in a systems approach for quarantine control of Hessian fly.