BBA - General Subjects (v.1573, #2)

Stabilization of S-adenosyl-l-methionine promoted by trehalose by Alessandra Morana; Paola Stiuso; Giovanni Colonna; Monica Lamberti; Maria Cartenı̀; Mario De Rosa (105-108).
S-adenosyl-l-methionine (SAM), an important metabolic intermediate of mammals, is a well-known therapeutic agent. The molecule is chemically unstable, both in solution and in dry state, and forms different degradation products. Because the chemical instability represents a real problem during the preparation of therapeutic formulations, we investigated the capacity of some sugars to improve the SAM stability over time. In the present work, we demonstrated that the disaccharide trehalose exercises a protective effect towards the lyophilized SAM slackening its degradation (65% of SAM was detected after 50 days at 37 °C). A parallel study, performed to stabilize the SAM into lyophilized yeast cells enriched in the sulfonium compound, assessed the positive effect of trehalose also in whole cells, but in lesser measure.
Keywords: S-adenosyl-l-methionine; Trehalose; Stability; Yeast;

Differential protection by nitroxides and hydroxylamines to radiation-induced and metal ion-catalyzed oxidative damage by Sandhya Xavier; Ken-ichi Yamada; Ayelet M Samuni; Amram Samuni; William DeGraff; Murali C Krishna; James B Mitchell (109-120).
Modulation of radiation- and metal ion-catalyzed oxidative-induced damage using plasmid DNA, genomic DNA, and cell survival, by three nitroxides and their corresponding hydroxylamines, were examined. The antioxidant property of each compound was independently determined by reacting supercoiled DNA with copper II/1,10-phenanthroline complex fueled by the products of hypoxanthine/xanthine oxidase (HX/XO) and noting the protective effect as assessed by agarose gel electrophoresis. The nitroxides and their corresponding hydroxylamines protected approximately to the same degree (33–47% relaxed form) when compared to 76.7% relaxed form in the absence of protectors. Likewise, protection by both the nitroxide and corresponding hydroxylamine were observed for Chinese hamster V79 cells exposed to hydrogen peroxide. In contrast, when plasmid DNA damage was induced by ionizing radiation (100 Gy), only nitroxides (10 mM) provide protection (32.4–38.5% relaxed form) when compared to radiation alone or in the presence of hydroxylamines (10 mM) (79.8% relaxed form). Nitroxide protection was concentration dependent. Radiation cell survival studies and DNA double-strand break (DBS) assessment (pulse field electrophoresis) showed that only the nitroxide protected or prevented damage, respectively. Collectively, the results show that nitroxides and hydroxylamines protect equally against the damage mediated by oxidants generated by the metal ion-catalyzed Haber–Weiss reaction, but only nitroxides protect against radiation damage, suggesting that nitroxides may more readily react with intermediate radical species produced by radiation than hydroxylamines.
Keywords: Radiation; Nitroxide; DNA damage; Protection;

Little information exists about single N-acetylglucosamine modifications on proteins in growth and developmental model systems. To explore these phenomena, Xenopus laevis oocytes from stages I–VI of oogenesis were isolated and proteins analyzed on SDS-PAGE. The proteins were probed with antibodies specific for O-GlcNAc. Levels of the O-GlcNAc protein modification were highest in stages I and II, while decreasing in stages III–VI. The reduction in amount of O-GlcNAc-modified proteins was correlated to increases in apparent O-GlcNAcase (streptozotocin-inhibitable neutral hexosaminidase), activity involved in removing protein monoglycosylations. The O-GlcNAc modification was also characterized during progesterone-stimulated oocyte maturation. Although O-GlcNAcase activity appeared relatively constant between quiescent and matured stage VI oocytes, a small decrease in the levels of both total and specific O-GlcNAc-modified proteins was observed. Investigating the function of O-GlcNAc during maturation, oocytes were incubated with compounds known to modulate the levels of the O-GlcNAc protein modification and then stimulated to mature. Oocytes treated with compounds known to increase O-glycosylation consistently matured slower than non-treated controls, while oocytes treated with compounds that decrease O-glycosylation matured slightly faster than controls. The O-GlcNAc modification may play important roles in both the developmental and cell division processes of X. laevis oocytes.
Keywords: Monoglycosylation; O-GlcNAc; Oocyte; Maturation; Oogenesis;

The in vivo and in vitro interactions of elastic and rigid vesicles with human skin by P Loan Honeywell-Nguyen; Anko M de Graaff; H.W Wouter Groenink; Joke A Bouwstra (130-140).
Elastic vesicles are the most novel development in vesicular systems design for dermal and transdermal drug delivery. However, interactions between these vesicles and human skin are not yet fully understood. In this study, the in vivo and in vitro interactions between elastic-, rigid vesicles and micelles with human skin were investigated. Vesicle and micelle solutions were applied onto human skin in vitro and in vivo. Subsequently, a series of tape strippings were performed, which were visualised by freeze fracture electron microscopy (FFEM). The results showed no ultrastructural changes in skin treated with rigid vesicles. Skin treated with elastic vesicles, however, showed a fast partitioning of intact vesicles into the deeper layers of the stratum corneum (SC), where they accumulated in channel-like regions. Only little vesicle material was found in the deepest layers of the SC, suggesting that the partitioning of intact vesicles from the SC into the viable epidermis is unlikely to happen. Treatment with micelles resulted in rough, irregular fracture planes. Similar results were obtained in vitro and in vivo, indicating an excellent in vitro/in vivo correlation. These results support the hypothesis that elastic vesicles have superior characteristics to rigid vesicles for the interaction with human skin. Elastic vesicles and micelles demonstrated very different interactions with human skin and hence probably also have different mechanisms of action for the enhancement of drug transport.
Keywords: Skin; Elastic vesicle; Tape stripping; Freeze fracture electron microscopy;

Non-catalytic role of carbonic anhydrase in rat intestinal absorption by Alan N Charney; Jesline Alexander-Chacko; Ramanashree Gummaconda; Richard W Egnor (141-148).
Carbonic anhydrase (CA) inhibition reduces NaCl absorption in rat distal ileum, a pH-sensitive, low CA activity tissue, and in distal colon, a CO2-sensitive, high CA activity tissue. We hypothesized that CA plays a non-catalytic role in NaCl absorption in these segments. Unidirectional fluxes of Na+ and Cl, and total HCO3 generation (estimated as the sum of radiolabeled HCO3 and CO2 produced from glucose) were measured in Ussing chambers in nominally CO2, HCO3 -free HEPES Ringer. Measurements were made in the presence and absence of 0.1 mM methazolamide, a membrane-permeant CA inhibitor. Ringer pH reduction from 7.6 to 7.1 stimulated ileal but not colonic Na+ and Cl absorption. In the ileum, methazolamide reduced J ms Na and J ms Cl and caused net Cl secretion at pH 7.6, and prevented the stimulatory effect of lowering pH. In the colon, methazolamide reduced Na+ and Cl absorption at pH 7.6. Total HCO3 generation was minimal in HEPES at pH 7.6 and 7.1 in both segments, was minimally affected by methazolamide, and did not account for the changes in Cl absorption caused by pH or methazolamide. We conclude that CA plays a role in ileal and colonic NaCl absorption independent of its catalytic function.
Keywords: Carbonic anhydrase; Rat; Ileum; Colon; Electrolyte transport;

Copper(II) complex of a tridentate ligand: an artificial metalloprotease for bovine serum albumin by H.Yamini Shrivastava; Mookandi Kanthimathi; Balachandran Unni Nair (149-155).
A copper(II) complex of 2, 6-bis(benzimidazo-2-yl) pyridine was synthesized and its binding properties with bovine serum albumin (BSA) has been evaluated. The binding plot obtained from the absorption titration data gives a binding constant of 2.4 (±0.3) ×103 M−1. It was found that the charge transfer band of the metal complex was perturbed in the presence of BSA. The gel electrophoresis pattern of BSA incubated with copper(II) complex shows the metalloproteolytic activity of the metal complex. In the presence of oxygen, protein undergoes site-specific cleavage by binding to the histidine residues of domain III, with the resultant formation of four fragments of molecular weight 49, 45, 22 and 17 kDa. This indicates the presence of two specific binding sites in the protein molecule. In the absence of molecular oxygen, the metal complex was found unable to cleave the protein. The circular dichroism (CD) spectrum of the isolated fragments shows nearly 38% and 32% of alpha helical content in 49 and 45 kDa fragments, respectively, which shows that the cleavage leads to no changes in the secondary structure of the protein fragments.
Keywords: Copper(II) complex; Molecular oxygen; Site-specific cleavage; Alpha helical content; Protein fragment;

Kinetic study on ESR signal decay of nitroxyl radicals, potent redox probes for in vivo ESR spectroscopy, caused by reactive oxygen species by Keizo Takeshita; Keita Saito; Jun-ichi Ueda; Kazunori Anzai; Toshihiko Ozawa (156-164).
The effect of the chemical structure of nitroxyl spin probes on the rate at which ESR signals are lost in the presence of reactive oxygen species (ROS) was examined. When the spin probes were reacted with either hydroxyl radical (OH) or superoxide anion radical (O2 •−) in the presence of cysteine or NADH, the probes lost ESR signal depending on both their ring structure and substituents. Pyrrolidine nitroxyl probes were relatively resistant to the signal decay caused by O2 •− with cysteine/NADH. Signal decay rates for these reactions correlated with reported redox potentials of the nitroxyl/oxoammonium couple of spin probes, suggesting that the signal decay mechanism in both cases involves the oxidation of a nitroxyl group. The apparent rate constants of the reactions between the spin probe and OH and between the spin probe and O2 •− in the presence of cysteine were estimated using mannitol and superoxide dismutase (SOD), respectively, as competitive standards. The rate constants for spin probes and OH were in the order of 109 M−1 s−1, much higher than those for the probes and O2 •− in the presence of cysteine (103–104 M−1 s−1). These basic data are useful for the measurement of OH and O2 •− in living animals by in vivo ESR spectroscopy.
Keywords: Reactive oxygen species; Nitroxyl radical; In vivo ESR spectroscopy; Reaction rate constant;

Environmental xenoestrogens have been implicated in human reproductive disorders and an increased incidence of breast cancer. Sulfation, a Phase II detoxification mechanism involving the cytosolic sulfotransferases (STs), may be an important mechanism in vivo for fending off these compounds. In this study, we report on the molecular cloning, expression, and purification of two human cytosolic STs, SULT2B1a and SULT2b1b. The activities of these two enzymes, as well as the other eight known human cytosolic STs previously prepared, toward representative environmental xenoestrogens were examined. Activity data showed that P-form (SULT1A1) PST displayed the highest activity toward these compounds, while SULT1C ST #2 also showed considerable activity, indicating that these enzymes may play a more important role in detoxification of environmental xenoestrogens. SULT1C ST #1, SULT2B1a ST, SULT2B1b ST and NST showed negligible or undetectable activity toward these compounds. The other four enzymes, M-form (SULT1A3) PST, SULT1B2 ST, SULT2A1 ST and SULT1E ST showed intermediate levels of activity toward some of these compounds. Kinetic studies on the sulfation of xenoestrogens by P-form (SULT1A1) PST were performed. The results are interpreted in the context of the endocrine-disrupting nature of these xenoestrogens.
Keywords: 3′-Phosphoadenosine 5′-phosphosulfate; Reverse transcriptase-polymerase chain reaction, sulfation; Sulfotransferase; Xenoestrogen;

Excretion of bisphenol A-glucuronide into the small intestine and deconjugation in the cecum of the rat by Hirokazu Sakamoto; Hiroshi Yokota; Ryoko Kibe; Yoshikatu Sayama; Akira Yuasa (171-176).
The environmental estrogen bisphenol A (BPA) is regarded as a modulator of endocrine systems and has been reported to have adverse effects on the reproductive organs of animals. In rats, BPA is metabolized to glucuronide by UDP-glucuronosyltransferase UGT2B1 in the liver and excreted into the bile. In the present study, we found that most of the bisphenol A-glucuronide (BPA-GA) excreted into the small intestine was deconjugated in the contents of the cecum. After BPA administration, BPA-GA was (immediately should be 15 min) found in the contents of the upper part of the small intestine, and then it moved to the lower part of the small intestine. However, only free BPA was found in the content of the cecum, and there was smaller amount of free BPA in the colon contents, indicating that BPA had been reabsorbed in the colon. BPA-GA was deconjugated by extract prepared from the cecum content which included highest β-glucuronidase (β-Gase) observed in Western blot analysis using antibodies against bacterial β-Gase.These results indicate enterohepatic circulation of BPA and suggest that the adverse effects of BPA are enhanced by repeated exposure.
Keywords: UDP-glucuronosyltransferase; Glucuronidation; Deconjugation; Bisphenol A; Digestive tract;

Extracellular metabolisation of NADH by blood cells correlates with intracellular ATP levels by Karl Nadlinger; Wilhelm Westerthaler; Danijela Storga-Tomic; Jörg G.D Birkmayer (177-182).
A new assay allowing quantitation of extracellular NADH metabolisation by intact blood cells was compared with the intracellular ATP/ADP ratio of these cells. The sensitivity, reproducibility and NADH specificity of this assay were determined. The diagnostic potential of this test was examined in a study with highly conditioned athletes. NADH consumption was measured before and immediately after maximum aerobic performance as well as 1 day later and was compared with the ATP/ADP level in these blood cells. A significant decline of cellular energy after aerobic performance was detected with both approaches to a similar extent (P<0.01). However, the extracellular NADH metabolisation assay (ENMA) is more convenient to perform than the determination of intracellular ATP/ADP. Due to its easy and versatile handling, a huge array of possible applications like monitoring the training efficiency of athletes, the fitness of senior citizens or the recovery from disease may be envisioned.
Keywords: NADH; ATP; Blood test; Sports;

Thyroid hormone (T3) is known to up-regulate the physiological expression of the Na+/K+ pump in cultured skeletal muscle. We recently reported that primary cultured rat skeletal muscle expresses only the α1, β1 and β2 protein isoforms of Na+/K+ pump. Interestingly, α2 mRNA is detectable while the α2 protein isoform is not. We therefore examined whether T3 might up-regulate the expression of Na+/K+ pump α2 isoform at the protein and mRNA level. We also examined the regulation by this hormone of the other isoforms of the pump. Primary cultures were treated with T3 for 48 h from day 4 to day 6 of differentiation. Protein and mRNA isoforms of Na+/K+ pump were identified by Western blotting and Northern blotting, respectively. T3 induced a marked increase in the β1 protein and a slight increase in the α1 protein. T3 did not affect expression of the β2 protein. The α2 protein was not detected in either untreated or T3-treated cells. In contrast, α2 mRNA was highly up-regulated by T3 treatment compared to the other isoforms. The lack of expression of the α2 protein isoform following T3 treatment suggests that posttranscriptional events related to this isoform may be dependent on other growth factors or hormones.
Keywords: Na+/K+ pump; Thyroid hormone; Skeletal muscle; Up-regulation;

Light-dependent induction of strongly increased microalgal growth by methanol by A Theodoridou; D Dörnemann; K Kotzabasis (189-198).
Low methanol concentrations (about 0.5% v/v) induce biomass production in cultures of the unicellular green alga Scenedesmus obliquus by more than 300%, compared to controls without this solvent. This effect on the microalgal growth was found to be dependent on the solvent concentration, the packed cell volume (PCV), light intensity and light quality. It could be shown that methanol addition leads to a decrease in size of the light harvesting complex (LHC) on the basis of chlorophylls and proteins, and thus to changes in structure and functioning of the photosynthetic apparatus. These alterations lead to enhanced photosynthesis and respiration rates. The action of methanol on the photosynthetic apparatus is thus comparable to the effect of enhanced CO2 concentrations. These findings support the previously proposed pathway for methanol metabolization with CO2 as the final product. We conclude that the subsequent assimilation of the increased CO2 amounts by the Calvin–Benson cycle is a possible explanation for the methanol-mediated increase in biomass production in terms of PCV. The methanol effect is observed only in the light and in the presence of a functioning photosynthetic apparatus. Preliminary action spectra suggest that the primary photoreceptor is a chlorophyll–protein complex with two absorption maxima at 680 and 430 nm, which may possibly be attributed to the reaction center of photosystem II (PSII).
Keywords: Biomass production; Methanol metabolization; Scenedesmus obliquus;