Improved coronary vascular function evoked by high-intensity treadmill training is maintained in arteries exposed to ischemia and reperfusion.

We hypothesized that myocardial contractile function and coronary arterial function are greater after ischemia and reperfusion in high-intensity treadmill-trained vs. sedentary rats. Rats performed 10 x 4-min bouts of treadmill running consisting of 2 min at 13 m/min + 2 min at 45-60 m/min (Etr) or were sedentary (Sed) for 12 wk. Animals then were instrumented to measure left ventricular (LV) contractility in response to three 15-min coronary occlusion (O) and 5-min reperfusion (R) cycles (Isc) or a sham operation (Sham). After the Isc and Sham protocols, hearts were excised and coronary arterial ( approximately 105 microm ID) function was evaluated by using isometric techniques. LV developed pressure, the first derivative of LV pressure at a developed pressure of 40 mmHg, and systolic blood pressure were not different between Etr (n = 14) and Sed (n = 7) rats before or after the Sham protocol. Furthermore, hemodynamic variables were similar in Etr (n = 14) and Sed (n = 13) animals before the Isc protocol and were depressed to the same degree by the three O-R cycles. Therefore, Etr did not alter myocardial contractile function in rats that were (i.e., Isc) or were not (i.e., Sham) exposed to ischemia and reperfusion. Acetylcholine-evoked relaxation (10-8 to 3 x 10-5 M) was greater (P < 0.05) in coronary arteries from Sham-Etr vs. Sham-Sed animals (5 of 8 doses tested) and Isc-Etr vs. Isc-Sed rats (3 of 8 doses tested). Maximal relaxation produced by sodium nitroprusside (10-4 M) was similar among groups. Vasocontractile responses produced by KCl (10-100 mM) and endothelin-1 (10-11-10-4 M) were greater (P < 0.05) in the presence vs. the absence of nitric oxide synthase inhibition (10-6 M NG-monomethyl-l-arginine) in vessels from Sham-Etr but not Sham-Sed rats and from Isc-Etr but not Isc-Sed rats. These findings suggest that Etr-evoked improvements in coronary function are maintained in small arteries even when exposed to ischemia and reperfusion.     

Effects of supplementary UV-B radiation on development of damping-off in spinach caused by the soil-borne fungusFusarium oxysporum

The effects of UV-B radiation (290–320 nm) on development of damping-off of spinach (Spinacia oleracea) caused by the fungusFusarium oxysporum were examined in a growth cabinet. The incidence of disease greatly increased when experimental plants were grown in visible radiation with supplementary UV-B radiation. This increase was suppressed by increasing the irradiation of visible radiation.Fusarium oxysporum was isolated from the roots of all damping-off plants and the roots of some unwilted plants, indicating that spinach infected with the pathogen did not necessarily suffer from damping-off in 15d. Supplementary UV-B radiation suppressed the increase in growth components such as the number of leaves, the plant height and the fresh weight of aboveground plant parts, but did not affect the fresh weight of roots. The ratio of the number of plants infected with pathogen to the total number of plants was over 80% irrespective of light conditions. It was suggested that the defense response of spinach to this pathogen was greatly influenced by the physiological state of aboveground plant parts resulting from supplementary UV-B radiation.     

Induction of a Nerve Growth Factor-Sensitive Kinase that Phosphorylates the DNA-Binding Domain of the Orphan Nuclear Receptor NGFI-B

Abstract: Nerve growth factor (NGF) induces the synthesis and the phosphorylation of the orphan nuclear receptor NGFI-B in PC12 cells. Previous work has shown that phosphorylation, by protein kinase A, of a specific serine in the DNA-binding domain inhibits its binding to the NGFI-B response element. Also, cytoplasmic extracts from PC12 cells phosphorylate this serine, and phosphorylation is greater in extracts from cells treated with NGF. The present work describes the induction, identification, and partial purification of a kinase (termed NGFI-B kinase I) from PC12 cell extracts that catalyzes this phosphorylation. Phosphorylation of the DNA-binding domain with this purified preparation inhibits its binding to the NGFI-B response element. The kinase is rapidly activated by treatment of the cells with NGF, and the activation lasts for at least several hours. It also is activated by fibroblast growth factor and epidermal growth factor (EGF), but the activation by EGF is quite transient. The kinase requires Mg²⁺ but will use Mn²⁺. The molecular mass of the kinase is 95–100 kDa, and it is different from protein kinase A, Fos kinase, or pp90 ^rsk . Comparison with a partially purified preparation of cyclic AMP response element-binding protein kinase, however, indicates that the two are either very similar or identical.     

An Estimate of Divergence Time of Parazoa and Eumetazoa and That of Cephalochordata and Vertebrata by Aldolase and Triose Phosphate Isomerase Clocks

Previously we suggested that four proteins including aldolase and triose phosphate isomerase (TPI) evolved with approximately constant rates over long periods covering the whole animal phyla. The constant rates of aldolase and TPI evolution were reexamined based on three different models for estimating evolutionary distances. It was shown that the evolutionary rates remain essentially unchanged in comparisons not only between different classes of vertebrates but also between vertebrates and arthropods and even between animals and plants, irrespective of the models used. Thus these enzymes might be useful molecular clocks for inferring divergence times of animal phyla. To know the divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata, the aldolase cDNAs from Ephydatia fluviatilis, a freshwater sponge, and the TPI cDNAs from Ephydatia fluviatilis and Branchiostoma belcheri, an amphioxus, have been cloned and sequenced. Comparisons of the deduced amino acid sequences of aldolase and TPI from the freshwater sponge with known sequences revealed that the Parazoa–Eumetazoa split occurred about 940 million years ago (Ma) as determined by the average of two proteins and three models. Similarly, the aldolase and TPI clocks suggest that vertebrates and amphioxus last shared a common ancestor around 700 Ma and they possibly diverged shortly after the divergence of deuterostomes and protostomes.     

Vibrio trachuri Sp. Nov., a New Species Isolated from Diseased Japanese Horse Mackerel

A new species, Vibrio trachuri sp. nov., was isolated from the cultured Japanese horse mackerel (Trachurus japonicus). These Vibrio were Gram negative, motile rods and formed yellow colonies on BTB teepol and TCBS plate, turned TSI medium to yellow and was sensitive to 150 μM O/129 (2,4-diamino-6,7-diisopropyl pteridine phosphate) like Listonella anguillarum which has been described as Vibrio anguillarum. However, the results of VP test and decarboxylation of lysine or dihydrolation of arginine suggested that these Vibrio are rather closely related to V. parahaemolyticus. DNA similarity determined by the microplate hybridization technique revealed that these Vibrio are genetically quite distant from Listonella anguillarum or V. parahaemolyticus and rather close to V. harveyi, although there was no Vibrio species which had more than 70% similarity value. From these results we propose to nominate Vibrio trachuri sp. nov. for this new Vibrio species.     

The lateral diffusion coefficient of lecithin molecules in single bilayer vesicles studied by 14N NMR

The ¹⁴N nuclear relaxation times T₁ and T₂ in egg yolk phosphatidylcholine have been observed in single bilayer vesicles dispersed in the media of different viscosities, ¹H₂O and ²H₂O. The lateral diffusion coefficient of lipid molecule D has been calculated according to the method reported earlier: D = 2.2 × 10^?8cm²s^?1 in ¹H₂O and 2.1 × 10^?8cm²s^?1 in ²H₂O at 20°C. They are in excellent agreement. This result gives a strong basis of usefulness of ¹⁴N NMR method in the evaluation of D without introducing any system perturbation.     

Chloride attachment negative-ion mass spectra of sugars by combined liquid chromatography and atmospheric pressure chemical ionization mass spectrometry

A method has been developed for the rapid determination of sugars, including molecular weight measurements, using high-performance liquid chromatography coupled with negative-ion, atmospheric-pressure chemical-ionization mass spectrometry. The chromatography was carried out on a 250 × 4 mm I.D. column packed with 7 μm NH₂-silica. The mobile phase consisted of a high percentage of methanol or acetonitrile with a small amount of chloroform. During the mass spectrometry, a strong base is formed from the polar solvent molecules by corona discharge, followed by ion—molecule reactions in the chemical ionization ion source (e.g. the methoxy anion is formed from methanol). This strong base reacts with the chloroform, generating chloride ions, which in turn react with the neutral sugar molecules as they elute from the chromatograph. The chloride ion and sugar interactions yield chloride-attachment ions, which are further stabilized by successive collisions. In this method, authentic monosaccharides and some oligosaccharides show dominant quasi-molecular ions, [M + Cl]⁻, with little fragmentation, and it is particularly useful for the molecular weight determination of sugars.     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

C-cell follicles of canine thyroid glands studied by PAS reaction and electron microscopy

Summary Small follicles composed solely of C cells were occasionally observed in large C cell groups of dog thyroid glands. The lumina of C-cell follicles were filled with, or contained peripheral depositions of PAS-positive amorphous material, which was similar in ultrastructural features to thyroglobulin-containing colloid in typical thyroid follicles. This indicates that C cells, in addition to secreting calcitonin, produce a glycoprotein that can be stored in the lumina of the follicles.     

α-Guanidinoglutaric Acid in Cobalt-Induced Epileptogenic Cerebral Cortex of Cats

: Guanidino compounds in the cobalt-induced epileptogenic cerebral cortex of cats were fluorometrically analysed by a JASCO G-520 guanidino compounds analyser, and an unknown high peak was observed in the chromatogram that was identical to the peak of authentic α-guanidinoglutaric acid. In another experiment, the substance was extracted from the cobalt focus tissue, converted into dimethylpyrimidyl derivative-butylester, and analysed by a GC/MS technique. The mass spectrum of the substance was identical to the dimethylpyrimidyl derivative of α-guanidinoglutaric acid butylester (M⁺= 365).