Nitric oxide-scavenging activity of polyhydroxylated fullerenol, C60(OH)24.

Investigation of the possible nitric oxide-scavenging activity of hydroxylated derivative of fullerene, fullerenol C60(OH)24, demonstrated that it expressed direct scavenging activity toward nitric oxide radical (NO) liberated within solution of sodium nitroprusside (SNP), a well known NO donor. In parallel, pre-treatment (30') with intratesticular injection of fullerenol (60 microg/each testis) prevented NO-induced decrease of catalase, glutathione transferase and glutathione peroxidase activities in the denucleated fraction of interstitial testicular cells of adult rats 2 h after intratesticular injection of SNP (20 microg/each testis). In addition, fullerenol decreased formation of thiobarbituric acid-reactive substances (TBA-RS) with similar efficiency as butylated hydroxy toluen (BHT), a well known antioxidant. Also, fullerenol expressed certain scavenging activity toward superoxide anion (O2-) in xanthine/xanthine oxidase system. In summary, results obtained in this study confirmed free radical-scavenging activity of fullerenol, and according to our knowledge, it is the first evidence of direct NO-quenching activity of hydroxylated C60 derivative in different milieu.     

Circadian ovulatory rhythms in Japanese quail: role of ocular and extraocular pacemakers

Previous studies have shown that the circadian system of Japanese quail is composed of multiple photic inputs and multiple oscillators. Among these are extraretinal photoreceptors that mediate both circadian and photoperiodic responses and circadian pacemakers in the eyes that, via neural and hormonal outputs, help to maintain rhythmicity of central circadian clocks (presumably located in the suprachiasmatic area of the hypothalamus). Furthermore, a component of the central circadian system is influenced by reproductive hormones. Under certain conditions, the circadian system of female quail can be induced to split into two circadian components: one driven by ocular pacemakers and one driven by feedback from reproductive hormones. Importantly, ovulation is either inhibited or permitted as these two oscillators (or sets of oscillators) constantly change internal phase relationships with each other, suggesting an "internal coincidence" mechanism in the control of ovulation. The oviposition patterns of quail in light-dark (LD) cycles also support an internal coincidence mechanism. The authors tested the hypothesis that the ocular pacemakers are an important component of an internal coincidence mechanism controlling ovulation by examinig the effects of blinding by complete eye removal (EX), and the effects of eye-patching, on the body temperature and oviposition patterns of quail exposed to 24-h LD cycles. They also examined the effects of EX on quail exposed to continuous light (LL) and to continuous darkness (DD). Neither EX nor eye-patching affected the oviposition patterns of birds in LD. Furthermore, robust body temperature and oviposition rhythms continued in EX birds in LL, but body temperature became arrhythmic in DD with the cessation of ovulation. The results do not show a role for ocular pacemakers in the control of ovulation, but they do support the hypotheses that (1) entrainment of the central oscillators by extraretinally perceived light is sufficient to preserve a normal ovulatory pattern in LD in the absence of the ocular pacemakers, and (2) in LL, feedback of reproductive hormones onto the central oscillators is sufficient to organize the circadian system even in the absence of the ocular pacemakers. Whether or not the ocular pacemakers are normally involved in the control of ovulation is still an open question.     

Effects of different dietary regimes alone or in combination with standardized Aronia melanocarpa extract supplementation on lipid and fatty acids profiles in rats

This study investigated different dietary strategies, high-fat (HFd), or standard diet (Sd) alone or in combination with standardized Aronia melanocarpa extract (SAE), as a polyphenol-rich diet, and their effects on lipids and fatty acids (FA) in rats with metabolic syndrome (MetS). Wistar albino rats were randomly divided into two groups: healthy and rats with MetS, and then depending on dietary patterns on six groups: healthy rats fed with Sd, healthy rats fed with Sd and SAE, rats with MetS fed with HFd, rats with MetS fed with HFd and SAE, rats with MetS fed with Sd, and rats with MetS fed with Sd and SAE. 4 weeks later, after an overnight fast (12–14 h), blood for determination of total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), index of lipid peroxidation (measured as TBARS), and FA was collected. Increased FA and lipid concentration found in MetS rats were reduced when changing dietary habits from HFd to Sd with or without SAE consumption. Consumption of SAE slightly affects the FA profiles, mostly palmitoleic acid in healthy rats and PUFA in MetS?+?HFd rats. Nevertheless, in a high-fat diet, SAE supplementation significantly decreases n-6/n-3 ratio, thereby decreasing systemic inflammation. Further researches are warranted to confirm these effects in humans.

     

Establishment of an EC50 database of pesticides using a Vibrio fischeri bioluminescence method

An EC₅₀ database was established to assess the acute toxicity of 16 PESTANAL pesticide standards and of seven pesticide commercial formulations using a Vibrio fischeri bioluminescence method. Half maximal effective concentration ( EC₅₀) is defined as the concentration of pollutant (in this case, pesticide) destroying 50% of the bacteria population and causing 50% bioluminescence inhibition, after a specified exposure time. Linear curves of bioluminescence inhibition versus pesticide concentration and EC₅₀ values were obtained for exposure times (t) of 5 or 15 min for these pesticides. The EC₅₀ values ranged from 6.90 × 10^?4 to 0.83 mg/ml (t = 5 min), and from 9.00 × 10^?4 to 0.37 mg/ml (t = 15 min) for pesticide standards, plus from 0.0077 to 0.74 mg/ml (t = 5 min), and from 0.0076 and 0.57 mg/ml (t = 15 min) for pesticide commercial formulations. The EC₅₀ database allowed classification of the pesticides under study into three categories according to their toxicity: very toxic, toxic and moderately toxic. These results demonstrated that the establishment of an EC₅₀ database and of linear curves of bioluminescence inhibition versus the pesticide concentration resulted in very important and irreplaceable tools to estimate the global and individual toxicity of pesticides present in environmental samples.     

Decreased expression of Slc26a4 (Pendrin) and Slc26a7 in the kidneys of carbonic anhydrase II-deficient mice

BACKGROUND/AIMS: Intercalated cells (ICs) of the kidney collecting duct are rich in carbonic anhydrase II (CAII), which facilitates proton and bicarbonate transport. Bicarbonate secretion is mediated via Pendrin (Slc26a4), which is expressed on the apical membrane of B-ICs and nonA-nonB ICs in the cortical collecting ducts (CCD). Bicarbonate absorption is mediated via anion exchanger 1 (AE1-Slc4a1) in the CCD and via AE1 and possibly Slc26a7 in the OMCD. Both exchangers are expressed on the basolateral membrane of A-ICs. The aim of this study was to examine the expression of pendrin, Slc26a7, and AE1 in the kidneys of CAII-deficient (CAR2-null) mice. METHODS: For the expression studies, we used real-time RT-PCR, Northern hybridization, immunolabeling, and immunoblotting. RESULTS: Pendrin mRNA expression was reduced 63% along with decreased pendrin immunolabeling in the cortex of CAR2-null mice present predominantly in nonA-nonB ICs. Slc26a7 mRNA expression was decreases by 73% and Slc26a7 immunolabeling, present in A-ICs, severely reduced in the outer medulla of CAR2-null mice. AE1 mRNA expression was decreased to a similar degree (62%) along with reduced AE1 immunolabeling. The expression of aquaporin 2 (AQP2) water channel, exclusively present in principal cells of the collecting duct, was comparable in the wild type and CAR2-null mice. CONCLUSION: CAII deficiency results in a significant decrease in the gene and protein expression of bicarbonate transport proteins from Slc26 gene family - Slc26a4 (pendrin) and Slc26a7. These results emphasize the critical role of CAII for the maintenance of the intercalated cell phenotype.     

Micropropagation of Pinus heldreichii

Micropropagation by organogenesis from mature embryos of Pinus heldreichii Christ. was achieved. The frequency of adventitious bud induction was higher on embryos grown on Gresshoff and Doy medium than on Von Arnold and Eriksson, or Murashige and Skoog medium. The greatest number of buds and developed shoots was obtained after induction with benzyladenine at 2.22 or 4.40 μM for four weeks. Shorter induction time was less effective for bud induction, but subsequent shoot elongation was accelerated. Shoots elongated on half-strength, growth regulator-free medium supplemented with activated charcoal. After pulse treatment with 1 mM indole-3-butyric acid twenty shoots were rooted, while agar-solidified medium supplemented with α-naphthaleneacetic acid (0.27 or 1.08 μM), or indole-3-butyric acid (0.25 or 0.98 μM) induced callus formation only. This revised version was published online in June 2006 with corrections to the Cover Date.     

Spermine Synthase Deficiency Leads to Deafness and a Profound Sensitivity to ��-Difluoromethylornithine

Male gyro (Gy) mice, which have an X chromosomal deletion inactivating the SpmS and Phex genes, were found to be profoundly hearing impaired. This defect was due to alteration in polyamine content due to the absence of spermine synthase, the product of the SpmS gene. It was reversed by breeding the Gy strain with CAG/SpmS mice, a transgenic line that ubiquitously expresses spermine synthase under the control of a composite cytomegalovirus-IE enhancer/chicken β-actin promoter. There was an almost complete loss of the endocochlear potential in the Gy mice, which parallels the hearing deficiency, and this was also reversed by the production of spermine from the spermine synthase transgene. Gy mice showed a striking toxic response to treatment with the ornithine decarboxylase inhibitor α-difluoromethylornithine (DFMO). Within 2–3 days of exposure to DFMO in the drinking water, the Gy mice suffered a catastrophic loss of motor function resulting in death within 5 days. This effect was due to an inability to maintain normal balance and was also prevented by the transgenic expression of spermine synthase. DFMO treatment of control mice or Gy-CAG/SpmS had no effect on balance. The loss of balance in Gy mice treated with DFMO was due to inhibition of polyamine synthesis because it was prevented by administration of putrescine. Our results are consistent with a critical role for polyamines in regulation of Kir channels that maintain the endocochlear potential and emphasize the importance of normal spermidine:spermine ratio in the hearing and balance functions of the inner ear.Polyamines are essential for viability in mammals. Knockouts of the genes for ornithine decarboxylase and S-adenosylmethionine decarboxylase, which are enzymes needed for the synthesis of putrescine, spermidine, and spermine, are lethal at early stages of embryonic development (1, 2). There is convincing evidence that the formation of hypusine in eIF5A, which requires spermidine as a precursor, is essential for eukaryotes (3). However, the function(s) of spermine is not so well established. Yeast mutants with inactivated spermine synthase grow at a normal rate (4). Mammalian cells in culture also grow normally in the presence of inhibitors of spermine synthase (5) or after inactivation of the spermine synthase gene (SpmS) (6–8). Inactivation of both of the genes that were originally described as encoding spermine synthases in plants leads to profound developmental defects (9–11), but recently it was discovered that one of these genes actually encodes a thermospermine synthase, and it appears that the lack of thermospermine may be responsible for these defects (12).In contrast, spermine is clearly required for normal development in mammals. The rare human Snyder-Robinson syndrome is caused by mutations in SpmS located in the X chromosome that drastically reduces the amount of spermine synthase (13, 14). This leads to mental retardation, hypotonia, cerebellar circuitry dysfunction, facial asymmetry, thin habitus, osteoporosis, and kyphoscoliosis. Male mice, which have an X chromosomal deletion that includes SpmS and have no detectable spermine synthase activity, do survive but are only viable on the B6C3H background (15–17). This mouse strain having an X-linked dominant mutation was isolated from a female offspring of an irradiated mouse and was termed gyro (Gy)² based on a circling behavior pattern in affected males (18). Subsequent studies have shown that the Gy mice have a deletion of part of the X chromosome that inactivates both Phex, a gene that regulates phosphate metabolism, and SpmS (16, 19). The lack of SpmS causes a total absence of spermine (6, 7, 15, 16). Such Gy mice suffer from hypophosphatemia, have a greatly reduced size, sterility, and neurological abnormalities, and have a short life span (6, 16, 18). All of these changes except the hypophosphatemia are reversed when spermine synthase activity is restored (20).The original characterization of Gy mice also reported preliminary indications that these mice had hearing defects lacking the Preyer reflex (21, 22). This is of particular interest in the context of polyamine metabolism because a drug, α-difluoromethylornithine (DFMO, Eflornithine), that targets ornithine decarboxylase has been shown to cause occasional hearing loss in some patients (23–26). Although DFMO was ineffective for cancer treatment, it is an extremely promising agent for cancer chemoprevention (27, 28). When combined with sulindac, DFMO treatment produced a substantial reduction in the recurrence of colorectal adenomas in a large clinical trial (27). DFMO is a major drug for the treatment of African sleeping sickness caused by Trypanosoma brucei (29, 30). It is also used as a topically applied cream for treatment of unwanted facial hair in women (31, 32). DFMO is generally well tolerated even at high doses, but reversible hearing loss has been reported in multiple clinical trials (25, 33), and a rarer irreversible defect has also been reported (34). These side effects are not observed at lower doses of DFMO (26, 27).Ototoxicity has been demonstrated to occur in experimental animals treated with DFMO including rats (35), guinea pigs (36), gerbils (37), and mice (38). Using immunohistochemistry, a high level of ornithine decarboxylase was observed in the inner ear of the rat, with the highest in the organ of Corti and lateral wall followed by the cochlear nerve (39). Measurements of polyamines in the relevant structures are very difficult due to the small amount of tissue available, but as expected, DFMO treatment reduced polyamine levels and ornithine decarboxylase activity in the inner ear of the guinea pig (36). A plausible explanation for the importance of polyamines in auditory physiology is based on their well documented role as regulators of potassium channels (38). The inward rectification of Kir channels is caused by blockage of the outward current by polyamines (40–42). Studies of the cloned mouse cochlear lateral wall-specific Kir4.1 channel showed that inward rectification was reduced and that there was a marked reduction in endocochlear potential (EP). It was proposed that DFMO treatment increases the outward Kir4.1 current, resulting in a drop in EP (38).In the experiments reported here, we have studied in more detail the role of polyamines in auditory physiology using Gy mice and crosses of these mice with transgenic CAG/SpmS mice (43). These mice express spermine synthase under the control of a composite cytomegalovirus-IE enhancer/chicken β-actin promoter, which was designed to provide ubiquitous expression (44–46). Assays of the spermine synthase activity in CAG/SpmS line 8 confirmed that there was a high level of expression of the transgene in many different organs and that this level was maintained for at least 1 year (43). Our studies confirm that Gy mice are totally deaf and that this condition is reversed by the expression of the SpmS gene. These changes are due to a virtually complete loss of the EP in the Gy mice. We have also examined the effect of DFMO on the Gy mice. Unexpectedly, it was found that these mice show a rapid and profound toxicity to this drug, leading to death within a few days. Within 5 days of exposure to DFMO in the drinking water, the DFMO-treated mice suffered a catastrophic loss of balance due to inner ear effects. This toxicity was also prevented by the transgenic expression of spermine synthase in the Gy background.