Effect of methylene methylimino linkage of antisense oligonucleotide to the platelet-derived growth factor A-chain on growth of vascular smooth muscle cells from spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells show exaggerated growth and increased expression of platelet-derived growth factor (PDGF) A-chain mRNA. We examined the effect of methylene methylimino linkage of antisense oligodeoxynucleotide, a novel modification of antisense oligodeoxynucleotide designed to increase nuclease resistance, to PDGF A-chain on the exaggerated growth of vascular smooth muscle cells from SHR. Methylene methylimino-linked oligodeoxynucleotide provided complete resistance against S1 nuclease. Methylene methylimino linkage of antisense oligodeoxynucleotide to PDGF A-chain resulted in a rapid inhibition of basal DNA synthesis of vascular smooth muscle cells from SHR. This inhibition was much greater than that produced by phosphorothioate linkage of antisense oligodeoxynucleotide to PDGF A-chain. The methylene methylimino linkage of antisense oligodeoxynucleotide to PDGF A-chain may prove useful in the treatment of arterial proliferative diseases including hypertension.

Structural origins of the exonuclease resistance of a zwitterionic RNA.

Nuclease resistance and RNA affinity are key criteria in the search for optimal antisense nucleic acid modifications, but the origins of the various levels of resistance to nuclease degradation conferred by chemical modification of DNA and RNA are currently not understood. The 2'-O-aminopropyl (AP)-RNA modification displays the highest nuclease resistance among all phosphodiester-based analogues and its RNA binding affinity surpasses that of phosphorothioate DNA by 1 degrees C per modified residue. We found that oligodeoxynucleotides containing AP-RNA residues at their 3' ends competitively inhibit the degradation of single-stranded DNA by the Escherichia coli Klenow fragment (KF) 3'-5' exonuclease and snake venom phosphodiesterase. To shed light on the origins of nuclease resistance brought about by the AP modification, we determined the crystal structure of an A-form DNA duplex with AP-RNA modifications at 1.6-A resolution. In addition, the crystal structures of complexes between short DNA fragments carrying AP-RNA modifications and wild-type KF were determined at resolutions between 2.2 and 3.0 A and compared with the structure of the complex between oligo(dT) and the D355A/E357A KF mutant. The structural models suggest that interference of the positively charged 2'-O-substituent with the metal ion binding site B of the exonuclease allows AP-RNA to effectively slow down degradation.

Studies in surgical trauma: oxidative stress in ischaemia-reperfusion of rat liver.

1. Hypovolaemic shock associated with surgical trauma has been studied in a rat liver ischaemia-reperfusion model by determination of oxidative stress, lipid peroxidation and tissue infiltration of polymorphonuclear leucocytes. 2. Liver ischaemia alone resulted in slight liver oedema and polymorphonuclear leucocyte infiltration, a slight increase in thiobarbituric acid-reacting substances (an index of lipid peroxidation) and decreases in liver reduced glutathione and total radical-trapping antioxidant parameter, indices of oxidative stress. Ischaemia plus 30 min of reperfusion further increased liver oedema, polymorphonuclear leucocyte infiltration and thiobarbituric-acid reacting substances, and further decreased liver reduced glutathione and total radical-trapping antioxidant parameter. 3. After 60 and 90 min of reperfusion, oedema (40% increase), polymorphonuclear leucocyte infiltration (40-fold increase) and thiobarbituric-acid reacting substances (20-fold increase) were maximal, and liver reduced glutathione (75-95% decrease) and total radical-trapping antioxidant parameter (85-90% decrease) were at a minimum. 4. All parameters were exacerbated by 24 h starvation. Liver reduced glutathione closely paralleled total radical-trapping antioxidant parameter, and ischaemia alone depleted both by 30% in fed rats and 50% in fasted rats. 5. Oxidative stress and lipid peroxidation were associated more with the period of reperfusion and polymorphonuclear leucocyte infiltration. Polymorphonuclear leucocyte infiltration into lung also occurred after 90 min of liver reperfusion. 6. Possible mechanisms of hepatic ischaemia-reperfusion-induced oxidative stress are discussed.

Role of tissue glutathione in prevention of surgical trauma.

1. Surgical trauma has been associated with pre-anaesthesia fasting, anaesthetic toxicity, haemorrhage, hypovolaemic shock, and other pathological phenomena. Tissue glutathione (GSH), thiobarbituric acid-reacting substances (TBAR), and radical-trapping activity (RTA) have been determined at various time intervals after fasting, anaesthesia, and also after hepatic ischaemia and reperfusion as a model for haemorrhage and hypovolaemic shock. 2. Light ether anaesthesia of rats resulted in an immediate (5 min) and progressive decrease in liver and kidney total glutathione (GSH and GSSG), which was much greater in animals that had been fasted for 20 h. TBARs, a measure of lipid peroxidation, in rat liver and kidney increased as total GSH decreased. Fasting (20 h) alone decreased tissue GSH by 50%, and increased TBAR 100%; fasting plus 30 min of ether anaesthesia decreased tissue glutathione by 80 to 85%, and increased TBAR by some 600%. 3. Liver ischaemia alone decreased total liver GSH by 20% in the fed rat, and 50% in the fasted rat. Ischaemia, followed by reperfusion, decreased liver total GSH by 70% in the fed rat, and 90% in the fasted rat. The ratio of GSH/GSSG decreased from 16 in control animals to 7 in the fasted ischaemic rat, then to 1 in the fasted, ischaemic rat reperfused for 90 min. RTA of liver closely paralleled liver total GSH levels. TBAR was increased by ischaemia alone (50-100%), but more (400%) by 90 min reperfusion. 4. A complex series of molecular mechanisms including: (1) GSH depletion; (2) induction of CYP2E1 activity; (3) generation of reactive oxygen species; (4) lipid peroxidation; (5) cytokine release; and (6) leucocyte activation, are advanced to account for the toxic phenomena of surgical trauma and multiple system organ failure.

The effects of ether anaesthesia on oxidative stress in rats--dose response.

Rats, with and without overnight fasting, were anaesthetised for 5, 15 and 30 min with diethyl ether, killed immediately and total glutathione (total GS), thiobarbituric acid-reacting substances (TBAR), radical-trapping activity (RTA), total cytochrome P450 (CYP), and 7-ethoxyresorufin O-deethylase (CYP1), 7-pentoxyresorufin O-dealkylase (CYP2B) and 4-nitrophenol hydroxylase (CYP2E1) activities of liver and kidney determined. Liver, after ether anaesthesia, but no fasting, showed 30-60% losses of total GS, RTA, and total CYP, after 5, 15 and 30 min of anaesthesia, while TBAR increased 10-, 20- and 35-fold for the same periods. Liver after ether anaesthesia and overnight fasting showed 50-85% losses of total GS, RTA and total CYP, for 0, 5, 15 and 30 min of anaesthesia, while TBAR increased 4-, 30-, 40- and 60-fold for the same periods of anaesthesia. Kidney changes were similar to those in liver. Liver CYP1 and CYP2B were decreased by 45% and 35%, respectively for 30 min of anaesthesia in fed rats, and by 80% and 30% respectively for 30 min of anaesthesia in fasted rats; in contrast, liver CYP2E1 was increased 30% by fasting alone and 70% by fasting plus 5 min of ether anaesthesia. Kidney CYP1 and CYP2B were similarly decreased by ether anaesthesia (70% and 50% respectively) in both fed and fasted rats, and CYP2E1 was similarly increased (by 40-90% in fed and 30-110% in fasted rats). The decrease in tissue total GS, RTA, total CYP, CYP1 and CYP2B, and the increase in lipid peroxidation products (TBAR), are all considered to be due to generation of reactive oxygen species and oxidative stress, associated with the increase in CYP2E1 activity that results from both fasting and exposure to diethyl ether.

Effects of ether anaesthesia and fasting on various cytochromes P450 of rat liver and kidney.

Fed and fasted, male, Wistar albino rats exposed to light ether anaesthesia and killed immediately or after 30 or 120 min recovery were compared with non-anaesthetized rats for changes in liver and kidney cytochrome P450 (CYP) activities. In fed rats, liver total CYP (nmol/mg protein) decreased by 30% immediately after ether, but was restored to normal levels after 30 min recovery; in fasted rats, liver total CYP increased by 20% by fasting alone, then decreased by 65% immediately after ether, and recovered to only 70% of control at 2 hr after ether. Rat liver cytochrome P4501A (CYP1A; 7-ethoxyresorufin O-deethylase or EROD activity) and cytochrome P4502B (CYP2B; 7-pentoxyresorufin O-dealkylase or PROD activity) were decreased after ether anaesthesia, similar to those for total CYP. In contrast, rat liver cytochrome P4502E1 (CYP2E1), determined by p-nitrophenol hydroxylation, increased by 40% by ether anaesthesia alone, 70% by fasting alone and 140% by ether plus fasting; these increases were confirmed by the CYP2E1-mediated activation of nitrosopyrrolidine and by immunoblot analysis using antibody to CYP2E1. In rat kidney, losses of total CYP, CYP1A and CYP2B, and increases of CYP2E1, induced by ether anaesthesia, were much more marked in fasted (90% loss in total CYP, 30% increase in CYP2E1) than in fed rats (slight loss in total cytochrome P450, 30% increase in CYP2E1). As maximum losses of total CYP in liver of fasted rats exposed to ether occurred at the time of maximum increase of CYP2E1 and maximum rate of generation of reactive oxygen species (ROS), it is suggested that the increase of CYP2E1, resulting from its stabilization by fasting and ether, leads to generation of ROS, increase in lipid peroxidation and consequent loss of total CYP, associated with the hepatic and renal necrosis seen in ether intoxication and surgical trauma.

Autoxidative injury with loss of cytochrome P-450 following acute exposure of rats to fasting and ether anaesthesia.

1. Exposure of fasted rats (20 h) to ether anaesthesia for 4 min resulted in increased exhalation of alkanes, an indication of lipid peroxidation in vivo. 2. Liver and kidney of the fasted rats anaesthetized with ether showed immediate 4-fold increases in luminol-amplified chemiluminescence, reaching maxima 30 min later, indicating the production of reactive oxygen species. 3. Liver and kidney cytosols of the fasted anaesthetized rats similarly showed immediate 4-fold increases of thiobarbituric acid-reactive material (malondialdehyde and other lipid peroxidation breakdown products) which attained maxima 60 min later. 4. Total cytochromes P-450 of liver and kidney of rats were decreased to 25-30% of control values after 20 h fasting and 4 min of ether anaesthesia, but were restored to normal levels 2 h later. Cytochrome P450 I (EROD activity) was decreased to 35-44% of control values by the ether anaesthesia and was restored to 80% of normal levels 2 h later. 5. Diether ether is known to be metabolized by cytochrome P450 IIE1 which is induced by fasting and by diethyl ether, and is possibly involved in the observed radical production, lipid peroxidation, and loss of cytochromes P-450.

Plasma prolactin concentrations in pinealectomized ewes receiving melatonin treatment and in pineal intact ewes maintained under a non-24-hour photoperiod.

We have investigated the profiles of prolactin secretion in relation to onset of breeding activity in Suffolk-Cross ewes with artificially modified melatonin rhythms. With treatments commencing in midsummer, groups of ewes were 1) subjected to a 8L:16D photoperiod, 2) maintained under a "short" (6L:16D) photoperiod repeated over a 22-h cycle that induces an elevation in plasma melatonin that does not endure for the entire dark phase, 3) pinealectomized (pnx) to abolish plasma melatonin levels, and 4) pinealectomized and treated with a melatonin implant (subcutaneous) to provide a constant (no 24-h rhythm) elevation in plasma melatonin. The onset of breeding activity was significantly advanced in both the 8L:16D and pnx/implant groups compared to the 6L:16D and untreated pnx ewes. The two latter groups displayed a normal timing in seasonal breeding activity. Low and high plasma prolactin levels corresponded with short and long photoperiods during both the 24 and 22-h cycles. There was no clearcut "seasonal" rhythm in plasma prolactin in either of the pnx groups. A clear differentiation was seen between reproductive response and prolactin response, particularly in the case of ewes monitored on 22-h cycles of short photoperiod.

Pineal and ovarian response to 22- and 24-h days in the ewe.

Melatonin secretion in ewes was entrained by 22-h light-dark cycles whether of long (16L:6D) or short (6L:16D) photoperiod. In photoperiods of 6L:16D, a phase-delay of melatonin secretion was evident, leading to a dark-phase duration shorter than that found in 8L:16D. Early onset of estrus was induced in anestrous ewes kept in 8L:16D, but not 6L:16D, from 22 July compared to controls in natural light. In photoperiods of 16L:6D, the melatonin profile corresponded precisely to the dark phase. Early offset of estrus was induced in estrous ewes kept in both 18L:6D and 16L:6D from 18 December compared to controls in natural light. Thus, when the duration of melatonin secretion was appropriate to the long photoperiod (16L:6D), but with a constantly changing phase position, a long-day reproductive response was found. Activity-rest cycles were not entrained by 16L:6D; thus the synchronization of melatonin and activity-rest cycles does not appear to be essential for the induction of a long-day reproductive response. These results support the hypothesis that the duration, not the circadian-phase position, of melatonin is critical to the induction of photoperiodic effects.

Use of an intraruminal soluble glass bolus containing melatonin for early lamb production.

The use of melatonin to advance the onset of seasonal oestrus is potentially useful for early lamb production. A number of delivery systems for melatonin have been developed and this study evaluates an intraruminal soluble glass bolus under field conditions. Anoestrous, non-lactating mule and Dorset cross mule ewes were treated in late June with two types of bolus of different solubilities (melatonin 1, 'slow release' and melatonin 2, 'extra slow release'). Rams were introduced in mid-August for a target lambing pattern in mid-January. The percentage pregnancy/lambing rates were 80/78, 97/92 and 100/100 in the control, melatonin 1 and melatonin 2 groups, respectively. The number of lambs born per ewe put to the ram was significantly increased by treatment with melatonin 2 (P less than 0.05) from 1.17 to 1.64 in the mule ewes and 1.64 to 1.72 in the Dorset cross mule ewes. The melatonin containing soluble glass bolus is a novel and convenient method of reducing the number of barren ewes in an early lambing flock.

How does melatonin control seasonal reproductive cycles?

The pineal gland is essential for the perception of photoperiod change in many species. Information about photoperiod length is conveyed through pineal secretion of the methoxyindole melatonin. Melatonin, suitably administered in physiological quantities is equipotent with artificial photoperiod in the induction of photoperiodic responses. Most experimental work suggests that it is the duration of high night time melatonin secretion (positively correlated with the length of the natural or artificial dark phase) which conveys the photoperiodic signal. Continuous release implants induce short day effects in ewes, entirely comparable to daily feeding of melatonin or short photoperiod. A minimum duration of secretion rather than a specific duration is therefore probably critical to short day effects. There appears to be a seasonal variation in sensitivity to short day melatonin effects (induction of early oestrus) which can be shifted to an earlier time of year following one oestrus advance the previous year. Short duration melatonin is read as a long day even secreted with 22 hour periodicity, suggesting a lack of circadian variation in sensitivity to melatonin.

Intraruminal soluble glass boluses containing melatonin can induce early onset of ovarian activity in ewes.

Soluble phosphate glass boluses (preparations A, B, and C) have been developed to release melatonin into the reticulo-rumen of ewes at relatively different (fast, medium or slow) rates. A fourth type (preparation D) containing no melatonin was used as a sham control. Four groups of seasonally anoestrous Suffolk-cross ewes were respectively dosed with preparations A, B, C or D on 4 July. Plasma samples were collected twice weekly for melatonin and progesterone assay. In Groups A, B and C, elevated daytime plasma concentrations of melatonin could be detected for about 5 weeks after bolus administration. However, the pattern of hormone release was variable between groups, with Group C animals maintaining higher plasma melatonin concentrations for a longer period. The control animals had undetectable daytime melatonin levels. The onset of cyclic ovarian activity in the animals treated with the 'slow' release bolus (Group C) was significantly (P less than 0.05) advanced compared to the control group. The 'fast' and 'medium' release treatments (Groups A and B) did not significantly alter the onset of ovarian activity. The results indicate the potential of a novel and convenient method of melatonin delivery for induction of early breeding activity in ewes.

Induction of early seasonal sensitivity to melatonin in Suffolk-Cross ewes.

Anoestrous Suffolk-Cross ewes can be induced into early seasonal ovarian activity by administration of melatonin at the appropriate time of day or by melatonin implants. This treatment is successful if commenced in June, but not earlier in April or May and suggests that a critical period of long days may be necessary before artificial short-day melatonin profiles act as winter time-cues. We have investigated whether the lack of sensitivity to melatonin in April could be overcome in ewes in which breeding activity had been artificially moved forward the previous season. The results indicate that this was indeed the case and that the breeding season in untreated ewes which also previously experienced an early induced breeding season reverted to the normal timing for the Suffolk-Cross breed.

Metabolism and pharmacokinetics of melatonin in the ewe.

The pineal hormone melatonin has been used to advance the onset of the breeding season in sheep and thus produce lambs earlier in the year. If this reproductive manipulation is to be used commercially, some knowledge of the route of metabolism and identity of possible metabolites is necessary. A major metabolite of melatonin in rodents and man is 6-hydroxymelatonin sulphate (acetyl-methoxytryptamine-6-sulphate [aMT6S]). No significant amounts of this metabolite could be found in the plasma of untreated ewes. After subcutaneous implantation of melatonin for 5 months, plasma levels of aMT6S were also insignificant. On the other hand, both a single oral dose of melatonin (3 mg) and daily oral dosing gave rise to circulating levels of aMT6S in the range of 150 to 1,500 pg/ml for at least 18 h. The profiles seen after 180 days treatment were similar to those seen after a single dose, indicating that this route of melatonin metabolism is not induced by chronic administration. Intravenous injection of melatonin (200 or 20 micrograms) gave rise to detectable levels of aMT6S in the plasma. These results indicate that the quantitative aspects of melatonin metabolism differ according to the route of administration.

Short-term variations of circulating melatonin in the ewe.

Melatonin levels have been studied in venous blood sampled at different frequencies (0.5-, 2-, and 60-min intervals) form intact ewes. All samples were taken during the dark phase of either natural or artificial photoperiods. In one experiment samples were taken simultaneously from both jugular veins to investigate the possible effects of "streaming" on the levels measured. Plasma cortisol was measured to ascertain whether or not the frequent removal of blood activated the ACTH stress axis. Plasma melatonin levels showed considerable variation with peaks of up to 365 pg/ml on a baseline of between 30 and 60 pg/ml. There was consistent evidence of intermittent peaks, the frequency of which increased with an increase in sampling frequency. Plasma cortisol showed no correlation with either the frequency or the amplitude of the melatonin peaks. When plasma samples were taken from both jugular veins a similar melatonin pattern was seen in the samples from both sides, but samples taken from the left jugular vein invariably showed higher levels than those taken from the right vein. This may be due to differential vascular drainage of the pineal to the two sides.

Changes in plasma concentrations of LH, FSH and prolactin in ewes receiving melatonin and short-photoperiod treatments to induce early onset of breeding activity.

Breeding activity was similarly advanced in ewes given continuous (s.c. implant) or timed (oral dose at 15.30 h) melatonin treatments or subjected to a short (8 h light: 16 h darkness) artificial photoperiod. Treatments commenced in mid-June and were terminated in mid-November. Weekly and serial blood samples were collected before and after treatments commenced, to ascertain the effects on plasma prolactin, LH and FSH concentrations. In addition, serial blood samples were collected for 24 h plasma prolactin and melatonin estimations before and after cessation of the treatments. Plasma prolactin levels were significantly reduced immediately following the start of the melatonin (implant and oral) and short-photoperiod treatments but 'rebounded' to levels greater than control values. The normal seasonal (spring) rise in plasma prolactin was noted in the following year. Before the onset of breeding activity, mean plasma LH and FSH concentrations and LH pulse frequency did not change following any of the treatments. The 24-h plasma melatonin profile accurately reflected the various applied treatments but had re-entrained to the prevailing (natural) photoperiod 1 week after termination of the treatments. There were no significant group differences in 24-h plasma prolactin levels 1 week before or 1 and 11 weeks after the treatments had ceased. Such treatments, although successfully advancing the onset of breeding activity and modifying the seasonal plasma prolactin rhythm, were not manifested through any apparent change in peripheral LH or FSH.

The ex vivo measurement of malondialdehyde and chemiluminescence as possible indices for anti-inflammatory drug evaluation.

A well-established model of foot-pad oedema in the rat, an example of a cell-mediated hypersensitivity reaction, was used in this study. Male rats were immunized on day 0 with Freund's complete adjuvant (FCA) and then challenged 6 days later with FCA in one hind paw. Within 4 h of the initiation of this acute inflammatory reaction, the presence of free radicals was detected as luminol-amplified chemiluminescence (LAC) directly from the inflamed paws. In addition, an increase in malondialdehyde (MDA), an end product of lipid peroxidation, was measured in both inflamed tissue and plasma. Groups of animals were treated with either diclofenac sodium, indomethacin, piroxicam or prednisolone either 1 h before or 6 h after challenge, oedema, LAC and MDA being measured at various times after challenge. The three non-steroidal anti-inflammatory drugs (NSAIDs) and prednisolone, given 1 h before challenge, significantly inhibited LAC and plasma MDA levels at 4 h post challenge. When given 6 h after challenge all drugs significantly inhibited oedema at 24 h. Furthermore, all the drugs inhibited tissue MDA and LAC but only indomethacin and piroxicam reached significance. The data suggest a role for lipid peroxidation and free-radical generation in inflammation and provide possible indices for in vivo drug activity and evaluation.

Free radical production at the site of an acute inflammatory reaction as measured by chemiluminescence.

A foot-pad oedema model was used to investigate the presence of free radicals using a chemiluminescence method. This model is an example of a cell mediated hypersensitivity reaction. Male rats were inoculated in the scruff with Freund's Complete Adjuvant (FCA) on Day 0 and then challenged 6 days later with FCA in one hind paw. An acute inflammatory reaction was initiated over the following 96 hours and within 4 hours of induction, reactive oxygen species were detected in the inflamed tissue. A peak of chemiluminescence activity was seen 8 hours after the induction of the inflammatory reaction, well before maximum oedema was observed. Using mannitol, catalase and DABCO to elucidate the nature of the reactive oxygen species it was found that hydroxyl radicals, hydrogen peroxide and singlet oxygen all contributed to this burst of oxidative activity and are therefore probably involved with the process of lipid peroxidation and the severity of an inflammatory reaction.