Isolation and structural elucidation of 4-(beta-D-glucopyranosyldisulfanyl)butyl glucosinolate from leaves of rocket salad (Eruca sativa L.) and its antioxidative activity

A structurally unique glucosinolate (GSL) was identified to be 4-(beta-D-glucopyranosyldisulfanyl)butyl GSL in rocket leaves. The positive-ion electrospray ionization mass spectrometry (ESI-MS) data indicated that the new GSL had a molecular weight of 521 (m/z 522, [M+H](+), as desulfo-GSL). The molecular formula of the substance was determined to be C(17)H(32)O(11)NS(3) (m/z 522.1143, [M+H](+)) based on its positive-ion high-resolution fast atom bombardment mass spectrometry (HR-FAB-MS) data. For the further confirmation, desulfated GSL of 4-(beta-D-glucopyranosyldisulfanyl)butyl GSL was prepared by commercial 1-thio-beta-D-glucose and dimeric 4-mercaptobutyl desulfo-GSL, which was also isolated from rocket leaves, and its chemical structure was then confirmed by MS data and nuclear magnetic resonance (NMR) spectroscopy. In addition, the antioxidative activity of 4-(beta-D-glucopyranosyldisulfanyl)butyl desulfo-GSL was measured by means of chemiluminescence (CL) for evaluating the functional properties. The antioxidative activity (2.089 unit/g) was relatively higher than that of dimeric 4-mercaptobutyl desulfo-GSL (1.227).     

Effects of nitric oxide synthase inhibitor on decrease in peripheral arterial stiffness with acute low-intensity aerobic exercise

We previously reported that even low-intensity, short-duration acute aerobic exercise decreases arterial stiffness. We aimed to test the hypothesis that the exercise-induced decrease in arterial stiffness is caused by the increased production of NO in vascular endothelium with exercise. Nine healthy men (age: approximately 22-28 yr) performed a 5-min single-leg cycling exercise (30 W) in the supine position under an intravenous infusion of NG-monomethyl-L-arginine (L-NMMA; 3 mg/kg during the initial 5 min and subsequent continuous infusion of 50 mug.kg(-1).min(-1) in saline) or vehicle (saline) in random order on separate days. The pulse wave velocity (PWV) from the femoral to posterior tibial artery was measured on both legs before and after the infusion at rest and 2 min after exercise. Under the control condition, exercised leg PWV significantly decreased after exercise (P <0.05), whereas nonexercised leg PWV did not show a significant change throughout the experiment. Under L-NMMA administration, exercised leg PWV was increased significantly by the infusion (P <0.05) but decreased significantly after the exercise (P <0.05). Nonexercised leg PWV increased with L-NMMA administration and maintained a significantly higher level during the administration compared with baseline (before the infusion, all P <0.05). The NO synthase blockade x time interaction on exercised leg PWV was not significant (P=0.706). These results suggest that increased production of NO is not a major factor in the decrease of regional arterial stiffness with low-intensity, short-duration aerobic exercise.     

Structural and functional differences in two cyclic bacteriocins with the same sequences produced by lactobacilli

Lactobacillus gasseri LA39 and L. reuteri LA6 isolated from feces of the same human infant were found to produce similar cyclic bacteriocins (named gassericin A and reutericin 6, respectively) that cannot be distinguished by molecular weights or primary amino acid sequences. However, reutericin 6 has a narrower spectrum than gassericin A. In this study, gassericin A inhibited the growth of L. reuteri LA6, but reutericin 6 did not inhibit the growth of L. gasseri LA39. Both bacteriocins caused potassium ion efflux from indicator cells and liposomes, but the amounts of efflux and patterns of action were different. Although circular dichroism spectra of purified bacteriocins revealed that both antibacterial peptides are composed mainly of alpha-helices, the spectra of the bacteriocins did not coincide. The results of D- and L-amino acid composition analysis showed that two residues and one residue of D-Ala were detected among 18 Ala residues of gassericin A and reutericin 6, respectively. These findings suggest that the different D-alanine contents of the bacteriocins may cause the differences in modes of action, amounts of potassium ion efflux, and secondary structures. This is the first report that characteristics of native bacteriocins produced by wild lactobacillus strains having the same structural genes are influenced by a difference in D-amino acid contents in the molecules.     

Age-related changes of Alzheimer's disease-associated proteins in cynomolgus monkey brains

We characterized senile plaques (SPs) immunohistochemically in cynomolgus monkey brains and also examined age-related biochemical changes of Alzheimer's disease (AD)-associated proteins in these brains from monkeys of various ages. In the neocortex of aged monkeys (>20 years old), we found SPs but no neurofibrillary tangles (NFTs). Antibodies against beta-amyloid precursor protein (APP) or apolipoprotein E (ApoE) stained SPs; however, the pattern of immunostaining was different for the two antigens. APP was present only in swollen neurites, but ApoE was present throughout all parts of SPs. Western blot analysis revealed that the pattern of APP expression changed with age. Although full-length APP695 protein was mainly expressed in brains from young monkeys (4-years-old), the expression of full-length APP751 protein was increased in brains from older monkeys (>20 years old). Biochemical analyses also showed that levels of various AD-associated proteins increased significantly with age in nerve ending fractions. Both SP-associated (APP) and NFT-associated proteins (tau, activated glycogen synthase kinase 3beta, cyclin dependent kinase 5, p35, and p25) accumulated in the nerve ending fraction with increasing age; however, we found no NFTs or paired helical filaments of tau in aged cynomolgus monkey brains. This age-related accumulation of these proteins in the nerve ending fraction was similar to that observed in our laboratory previously for presenilin-1 (PS-1). The accumulation of these SP-associated proteins in this fraction may be a causal event in the spontaneous formation of SPs; thus, SPs may be formed initially in nerve endings. Taken together, these results suggest that intensive investigation of age-related changes in the nerve ending and in axonal transport will contribute to a better understanding of the pathogenesis of neurodegenerative disorders such as AD.     

Bone-marrow-derived myofibroblasts contribute to the cancer-induced stromal reaction

To confirm whether human cancer-induced stromal cells are derived from bone marrow, bone marrow (BM) cells obtained from beta-galactosidase transgenic and recombination activating gene 1 (RAG-1) deficient double-mutant mice (H-2b) were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice (H-2d). The human pancreatic cancer cell line Capan-1 was subcutaneously xenotransplanted into SCID recipients and stromal formation was analyzed on day 14 and on day 28. Immunohistochemical and immunofluorescence studies revealed that BM-derived endothelial cells (X-gal/CD31 or H-2b/CD31 double-positive cells) and myofibroblasts (X-gal/alpha-smooth muscle actin or H-2b/alpha-smooth muscle actin double-positive cells) were present within and around the cancer nests. On day 14, the frequencies of BM-derived endothelial cells and BM-derived myofibroblasts were 25.3+/-4.4% and 12.7+/-9.6%, respectively. On day 28, the frequency of BM-derived endothelial cells was 26.7+/-9.7%, which was similar to the value on day 14. However, the frequency of BM-derived myofibroblasts was significantly higher (39.8+/-17.1%) on day 28 than on day 14 (P<0.05). The topoisomerase IIalpha-positive ratio was 2.2+/-1.2% for the H-2b-positive myofibroblasts, as opposed to only 0.3+/-0.4% for the H-2b-negative myofibroblasts, significant proliferative activity was observed in the BM-derived myofibroblasts (P<0.05). Our results indicate that BM-derived myofibroblasts become a major component of cancer-induced stromal cells in the later stage of tumor development.     

Characterization of two different 2-oxoglutarate:ferredoxin oxidoreductases from Hydrogenobacter thermophilus TK-6

A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, fixes carbon dioxide via the reductive TCA cycle. 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) is one of the key enzymes of this cycle. Strain TK-6 has two distinct OGOR enzymes termed For and Kor. These enzymes were purified and characterized following heterologous expression in Escherichia coli. The specific activity of For was approximately one-tenth of that of Kor. Additionally, For showed higher thermo-stability than Kor under both aerobic and anaerobic conditions. Western blot analysis showed that both of For and Kor were expressed when strain TK-6 was grown under aerobic conditions. In contrast, only Kor was expressed when the strain was grown under anaerobic conditions using nitrate as a terminal electron acceptor. These results indicate that For supports the optimal growth of strain TK-6 in the presence of oxygen.     

Changes of low-frequency vibrational modes induced by universal 15N- and 13C-isotope labeling in S2/S1 FTIR difference spectrum of oxygen-evolving complex

The effects of universal (15)N- and (13)C-isotope labeling on the low- (650-350 cm(-1)) and mid-frequency (1800-1200 cm(-1)) S(2)/S(1) Fourier transform infrared (FTIR) difference spectrum of the photosynthetic oxygen-evolving complex (OEC) were investigated in histidine-tagged photosystem (PS) II core particles from Synechocystis sp. PCC 6803. In the mid-frequency region, the amide II modes were predominantly affected by (15)N-labeling, whereas, in addition to the amide II, the amide I and carboxylate modes were markedly affected by (13)C-labeling. In the low-frequency region, by comparing a light-induced spectrum in the presence of ferricyanide as the electron acceptor, with the double difference S(2)/S(1) spectrum obtained by subtracting the Q(A)(-)/Q(A) from the S(2)Q(A)(-)/S(1)Q(A) spectrum, considerable numbers of bands found in the light-induced spectrum were assigned to the S(2)/S(1) vibrational modes in the unlabeled PS II core particles. Upon (13)C-labeling, changes were observed for most of the prominent bands in the S(2)/S(1) spectrum. Although (15)N-labeling also induced changes similar to those by (13)C-labeling, the bands at 616(-), 605(+), 561(+), 555(-), and 544(-) cm(-1) were scarcely affected by (15)N-labeling. These results indicated that most of the vibrational modes found in the low-frequency spectrum are derived from the coupling between the Mn-cluster and groups containing nitrogen and/or carbon atom(s) in a direct manner and/or through hydrogen bonding. Interestingly, an intensive band at 577(-) cm(-1) was not affected by (15)N- and (13)C-isotope labeling, indicating that this band arises from the mode that does not include either nitrogen or carbon atoms, such as the skeletal vibration of the Mn-cluster or stretching vibrational modes of the Mn-ligand.     

Direct interaction between substrates and endogenous steroids in the active site may change the activity of cytochrome P450 3A4

CYP3A4 exhibits unusual kinetic characteristics that result from the metabolism of multiple substrate including endogenous steroids and some drugs that coexist at the active site. To clarify the mechanism of the effect of endogenous steroids on the drug metabolism, the interaction between substrates, nevirapine (NVP) and carbamazepine (CBZ), and endogenous steroids was investigated by theoretical calculations. When the activities of NVP 2-hydroxylation and CBZ 10,11-epoxidation by expressed CYP3A4 were measured in the presence of steroids, NVP 2-hydroxylation was found to be remarkably increased by aldosterone and inhibited by estradiol. CBZ 10,11-epoxidation was increased by androstenedione. Three-dimensional computer modeling has shown that the active site of CYP3A4 is especially large, permitting access of two substrate molecules. The interactions between NVP and aldosterone and between CBZ and androstenedione were estimated by theoretical calculations assuming the substrate and steroids to be present in the active site at the same time. It was shown that NVP or CBZ would be stably fixed close to the oxygen atom at the sixth ligand of heme by interaction with steroids, suggesting that NVP and CBZ may be hydroxylated more easily due to the interaction with steroids. Estradiol was also expected to interact with NVP via a pi/pi interaction between a benzene ring, in which the NVP hydroxylation site is located, and a benzene ring of estradiol, suggested to inhibit the reaction. From these results, interactions between the substrate and endogenous steroids in the active site may change the activity of CYP3A4.     

Hippocampal cytochrome P450s synthesize brain neurosteroids which are paracrine neuromodulators of synaptic signal transduction

Hippocampal pyramidal neurons and granule neurons of adult male rats are equipped with a complete machinery for the synthesis of pregnenolone, dehydroepiandrosterone, 17beta-estradiol and testosterone as well as their sulfate esters. These brain neurosteroids are synthesized by cytochrome P450s (P450scc, P45017alpha and P450arom) from endogenous cholesterol. Synthesis is acutely dependent on the Ca(2+) influx attendant upon neuron-neuron communication via N-methyl-D-aspartate (NMDA) receptors. Pregnenolone sulfate, estradiol and corticosterone rapidly modulate neuronal signal transduction and the induction of long-term potentiation via NMDA receptors and putative membrane steroid receptors. Brain neurosteroids are therefore promising neuromodulators that may either activate or inactivate neuron-neuron communication, thereby mediating learning and memory in the hippocampus.