Chk2 activation dependence on Nbs1 after DNA damage

The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G, arrest. Here we show that the ATM-dependent activation of Chk2 by gamma- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells, interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1, Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.

A rheology study of high-energy radiolysis of a semicrystalline ethylene-propylene copolymer containing DOP mobilizer

The ractiolysis of a poly(ethylene-co-propylene), Elpro grade P 750 J, marketed by Thai Polypropylene Co. Ltd. for the manufacture of medical goods, was investigated at ambient temperature and melt rheology measured. The roles of calcium stearate, blended with the Elpro as a processing aid, and dioctyl phthalate (DOP), added in various amounts as a radical scavenger, were assessed. Following radiolysis, G' and the viscosity of the polymer melts at 453 K both decreased with increasing radiation dose, even when the mobilizer was present. The results indicated that although the DOP did scavenge radicals, it did not protect the polymer from net chain scission in a low-dose regimen. The value of (G(S) - 4G(X)) was approximately 0.6-0.7. (c) 2006 Wiley Periodicals, Inc.

Methadone maintenance treatment reduces heroin injection in New South Wales prisons

The purpose of this study was to examine whether methadone maintenance treatment reduces injecting risk behaviour land therefore transmission of blood-borne viral infections) among prisoners in New South Wales (NSW), using comparison of retrospective reports of drug use in prisons for people who received standard drug treatment, time-limited methadone treatment and methadone maintenance treatment, The setting for the study was the NSW prison system. One hundred and eighty-five injecting drug users who had been recently released from NSW prisons were recruited in 1993, Self-reported drug use and injecting risk behaviour were compared in inmates who received standard drug treatment (counselling), time-limited methadone treatment and methadone maintenance treatment. HIV status was determined by serology, Intervention comprised high and low dose methadone treatment and counselling. The groups were similar in terms of most basic demographic characteristics but subjects who had been maintained on methadone reported a significantly lower prevalence of heroin injection, syringe sharing and scored lower on an HIV Risk-taking Behavioural Scale than subjects who received standard drug treatment and time-limited methadone treatment, This study suggests that methadone treatment is associated with reduced injecting risk behaviour in prison with adequate (greater than 60 mg) dose and duration in treatment. These treatment conditions are known to increase effectiveness in community-based methadone programmes. Prospective studies are required to evaluate the effectiveness of methadone programmes in the prevention of HIV and other blood-borne viral infections among IDU prisoners.

Brain region-specific studies of the excitatory behavioral effects of morphine-3-glucuronide

This study was designed to determine in rats whether morphine-3-glucuronide (M3G) produces its neuro-excitatory effects most potently in the ventral hippocampus (as has been reported previously for subanalgesic doses of opioid peptides). Guide cannulae were implanted into one of seven regions of the rat brain: lateral ventricle; ventral, CA1 and CA2-CA3 regions of the hippocampus; amygdala; striatum or cortex. After a 7 day recovery period, rats received intracerebral injections of (i) M3G (1.1 or 11 nmol) (ii) DADLE ([D-Ala(2),D-Leu(5)]enkephalin), (45 nmol, positive controls) or (iii) vehicle (deionised water), and behavioral excitation was quantified over 80 min. High-dose M3G (11 nmol) evoked behavioral excitation in all brain regions but the onset, severity and duration of these effects varied considerably among brain regions. By contrast, low-dose M3G (1.1 nmol) evoked excitatory behaviors only when administered into the ventral hippocampus and the amygdala, with the most potent effects being observed in the ventral hippocampus. Prior administration of the nonselective opioid antagonists, naloxone and beta-funaltrexamine into the ventral hippocampus, markedly attenuated low-dose M3G's excitatory effects but did not significantly alter levels of excitation evoked by high-dose M3G. Naloxone given 10 min after M3G (1.1 or 11 nmol) did not significantly attenuate behavioral excitation. Thus, M3G's excitatory behavioral effects occur most potently in the ventral hippocampus as reported previously for subanalgesic doses of opioid peptides, and appear to be mediated through at least two mechanisms, one possibly involving excitatory opioid receptors and the other, non-opioid receptors.

Intermittent heparinised saline flushes for maintaining indwelling peripheral and central intravenous catheters in diabetic dogs

Intermittent low-dose heparinised saline flushes were found to be efficacious for maintaining patency of indwelling peripheral and central intravenous catheters in diabetic dogs. The catheters were flushed with 1 mL of 1 U/mL heparinised saline every two hours immediately following blood sample collection, or every 12 hours when not being used for sampling. Central catheters were flushed with saline solution first to clear the line before instillation of the heparinised saline. Patency of 54/57 (95%) of the peripheral catheters and 30/32 (94%) of the central catheters was achieved for up to 36 hours and five days, respectively. No phlebitis, or local or systemic infections were observed and, in each case, catheter failure was attributable to obstruction or extravasation. It is unlikely that there will be any contraindications to this flushing technique and its introduction may improve intravenous catheter survival and reduce catheter-associated complications in hospitalised dogs.

Use of methotrexate in older patients - A risk-benefit assessment

Methotrexate is eliminated almost entirely by the kidneys. The risk of methotrexate toxicity is therefore increased in patients with poor renal function, most likely as a result of drug accumulation. Declining renal function with age may thus be an important predictor of toxicity to methotrexate. Up to 60% of all patients who receive methotrexate for rheumatoid arthritis (RA) discontinue taking it because of adverse effects, most of which occur during the first year of therapy. Gastrointestinal complications are the most common adverse effects of methotrexate, but hepatotoxicity, haematological toxicity, pulmonary toxicity, lymphoproliferative disorders and exacerbation of rheumatic nodules have all been reported, Decreased renal function as a result of disease and/or aging appears to be an important determinant of hepatic, lymphoproli ferative and haematological toxicity, Concomitant use of low doses of folic acid has been recommended as an approach to limiting toxicity. Interactions between methotrexate and several nonsteroidal anti-inflammatory drugs have been reported, but they may not be clinically significant. However, caution is advised in the use of such combinations in patients with reduced renal function. More serious toxicities (e.g. pancytopenia) may result when other inhibitors of folate utilisation [e.g. cotrimoxazole (trimethoprim-sulfamethoxazole)] or inhibitors of renal tubular secretion (e.g. probenecid) are combined with methotrexate. Before starting low dose methotrexate therapy in patients with RA, a full blood count, liver function tests, renal function tests and chest radiography should be performed. Blood counts and liver function tests should be repeated at regular intervals. Therapeutic drug monitoring of methotrexate has also been suggested as a means of limiting toxicity. Patients with RA usually respond very favourably to low dose methotrexate therapy, and the probability of patients continuing their treatment beyond 5 years is greater than for other slow-acting antirheumatic drugs. Thus, given its sustained clinical utility and relatively predictable toxicity profile, low dose methotrexate is a useful addition to the therapy of RA.

Short-term and long-term cardiovascular actions of different doses of perindopril in the rabbit

Short-term (one week) and chronic (six week) cardiovascular effects of orally administered perindopril were examined in the rabbit to demonstrate if short-term results can predict chronic outcomes. In short-term treatment, five doses of perindopril were examined in random order separated by a one week recovery period in each of six rabbits. Two doses of perindopril which resulted in a moderate hypotensive effect (-14 mmHg) and no hypotensive effect, respectively, were then selected for long-term treatment. Each rabbit in the short-term study received perindopril in doses of 0.01, 0.06, 0.32, 1.8 and 10 mg kg(-1) day(-1) for a week at a time. Rabbits on long-term treatment received either 0.3 or 0.01 mg kg(-1) day(-1) perindopril for six weeks. All rabbits had their mean arterial blood pressure (MAP) and heart rate recorded throughout treatment. Plasma angiotensin I (AngI), perindoprilat, angiotensin converting enzyme (ACE) inhibition were also assayed. Perindopril treatment for one week produced a dose-dependent hypotensive effect with the threshold dose, 0.06 mg kg(-1) day(-1), producing a 6.5+/-1.8 mmHg fall in MAP. The highest dose (10.0 mg kg(-1) day(-1)) produced a large fall in blood pressure of -29.6+/-4.2 mmHg. The 0.01 and 0.06 mg kg(-1) day(-1) doses of perindopril produced an average 2.65 fold increase in plasma AngI levels compared to the initial control. The three higher doses (0.32-10.0 mg kg(-1) day(-1)) of perindopril produced an equivalent 5.7 fold increase in plasma AngI levels compared to the initial controls. However, over six weeks 0.01 mg kg(-1) day(-1) perindopril induced a similar decrease in MAP as the 30 fold higher dose (-9.3 mmHg compared to -11.7 mmHg,). This was in spite of a 3 fold difference in plasma perindoprilat concentrations between the high and low dose perindopril groups. Plasma ACE inhibition was >80% with both doses of perindopril. The results indicate that while perindopril decreases MAP in a dose-dependent manner in short-term (one week) periods, over longer treatment times (six weeks) low concentrations of perindopril, non-hypotensive with shortterm treatment, may be as anti-hypertensive as considerably higher doses. (C) 1996 The Italian Pharmacological Society.

Intravenous cisplatin administration in sulphur-crested cockatoos (Cacatua galerita): Clinical and pathologic observations

The prevalence of neoplasia in birds is generally low; however, in some species of companion and aviary birds, the incidence is high and neoplasia is a common cause of death. Surgical excision or limb amputation has been performed as the therapeutic plan. Chemotherapy in the treatment of avian neoplasia is largely empirical and poorly documented. For example, cisplatin has been used intralesionally in macaws (Ara species) with limited clinical success. Eight sulphur-crested cockatoos (Cacatua galerita), under general isoflurane anesthesia, were infused intravenously with cisplatin at 6.4 or 1.0 mg/kg over 1 hour and hydrated with lactated Ringer's solution for 1 hour before and 2 hours after cisplatin infusion. Birds were euthanatized 96 hours after infusion, except for 2 birds given the low cisplatin dose, which were euthanatized on day 35 after dosing. All birds tolerated the study procedure while under anesthesia. Blood pressure, heart rate, and respiratory rate did not change significantly. In the low-dose group, the mean cloacal temperature decreased significantly during the infusion period (P < .001) and then rose progressively to preinfusion values by 24 hours. Also in this group, the mean body weight tended to increase during the infusion period before significantly decreasing (P < .05) by 5% at 96 hours after dosing. At 24 hours after dosing, all birds were bright and eating. However, intermittent regurgitation and fecal changes (moist, dark green feces and yellow urates) occurred in 3 of 8 birds, especially those given the high dose. By 72 hours after dosing, droppings in the low-dose group were normal in appearance. One bird in the high-dose group died by 94 hours after dosing. Myelosuppression was not observed in any bird and at necropsy, no evidence of cisplatin toxicity was found except in 1 bird given the high cisplatin dose. On histology, this bird showed nephrotoxicity, and its serum uric acid levels and mean estimated white blood cell count increased significantly by 24 hours after dosing. This paper reports for the first time the effect of systemic cisplatin administration in birds and provides veterinarians data for formulating efficacious and safe protocols for platinum-containing compounds when treating neoplasia in parrots and other companion birds.

Growth hormone receptor and IGF-1 receptor immunoreactivity during orthodontic tooth movement in the prednisolone-treated rat

Bone remodeling during tooth movement is regulated by local and systemic factors. Two regulators of bone metabolism are growth hormone (GH) and insulin-like growth factor-I (IGF-1). Their effects are mediated via binding to GH receptor (GHR) and IGF-I receptor (IGF-IR) in target tissues. Corticosteroids may affect the activity of these growth factors. This study examined the effect of prednisolone on GHR and IGF-IR expression in dental tissues following orthodontic tooth movement. The corti ticosteroid-treated group (N = 6) was administered prednisolone ( 1 mg/kg,) daily and the control group (N = 6) received equivalent volumes of saline. An orthodontic force (30 g) was applied to the maxillary first molar. Animals were sacrificed 12 days postappliance insertion. Sagittal sections of the first molar were stained for GHR and IGF-IR immunoreactivity. GHR and IGF-IR cell counts were elevated following appliance-treatment. Orthodontic tooth movement appeared to up-regulate GHR and IGF-IR immunoreactivity, but this up-regulation was reduced following prednisolone treatment. The suppression of GHR and IGF-I immunoreactivity in steroid-treated animals infers the mechanism whereby bone resorption and deposition, necessary for orthodontic tooth movement, may be inhibited by prednisolone. However, at 12 days postappliance insertion. no difference in orthodontic tooth movement was observed following low-dose prednisolone treatment.

Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice

Type I diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta -cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta -cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in A-PNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta -cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(-/-) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.

Use of cyclic variation of integrated backscatter to assess contractile reserve and myocardial viability in chronic ischemic left ventricular dysfunction

The detection of viable myocardium has important implications for management, but use of stress echocardiography to detect this is subjective and requires exposure to dobutamine. We investigated whether cyclic variation (CV) of integrated backscatter (IB) from the apical views could provide a resting study for detection of contractile reserve (CR) and prediction of myocardial viability in 27 patients with chronic ischemic left ventricular (LV) dysfunction. Repeat echocardiography was performed after 6.7 +/- 3.8 months of follow-up; 14 patients underwent revascularization and 13 were treated medically. Using a standardized dobutamine echocardiography (DbE) protocol, images from three apical views were acquired at 80-120 frames/sec at rest and during stress. CR was identified if improvement of wall motion was observed at low dose (5 or 10 mug/kg/min) DbE. Myocardial viability was characterized by improvement at follow-up echocardiography in patients with revascularization. CVIB at rest and low dose dobutamine were assessed in 194 segments with resting asynergy (severe hypokinesis or akinesis), of which 88 (45%) were in patients who underwent revascularization. Of these, CVIB could be measured in 190 (98%) segments at rest and 185 (95%) at low dose dobutamine. Sixty-two (33%) segments had CR during low dose DbE and 50 (57%) segments showed wall-motion recovery (myocardial viability) at follow-up echocardiography. Segments with CR had significantly higher CVIB at rest (P < 0.001) and low dose dobutamine (P = 0.005) than segments without CR. Using optimal thresholds of CVIB (> 8.2 dB) at rest, the accuracy of CVIB for detecting CR was 70%. Compared with nonviable segments, viable segments had significantly higher CVIB at rest (P < 0.001) and low dose dobutamine (P < 0.001). Using optimal thresholds of CVIB (> 5.3 dB) at rest, the accuracy of CVIB for detecting myocardial viability was 85%, which was higher than that in conventional DbE (62%, P < 0.01). Thus, assessment of CV.TB from the apical views is a feasible and accurate tool for detecting CR and predicting myocardial viability in chronic LV dysfunction.

Use of GnRH agonist implants for long-term suppression of fertility in extensively managed heifers and cows

The ability of gonadotrophin releasing hormone (GnRH) agonist implants to suppress ovarian activity and prevent pregnancies, long-term, was examined in heifers and cows maintained under extensive management. At three cattle stations, heifers (2-year-old) and older cows (3- to 16-year-old) were assigned to a control group that received no treatment, or were treated with high-dose (12 mg, Station A) or low-dose (8 mg, Station B and Station Q GnRH agonist implants. The respective numbers of control and GnRH agonist-treated animals (heifers + cows) at each station were: Station A, 20 and 99; Station B, 19 and 89; Station C, 20 and 76. Animals were maintained with 4% bulls and monitored for pregnancy at 2-monthly intervals for approximately 12 months. Pregnancy rates for control heifers and control cows ranged from 60-90% and 80-100%, respectively, depending on the study site. The respective number of animals (heifers + cows) treated with GnRH agonist that conceived, and days to first conception, were: Station A, 9 (9%) and 336 3 days; Station B, 8 (10%) and 244 +/- 13 days; Station C, 20 (26%) and 231 +/- 3 days. Treatment with high-dose GnRH agonist prevented pregnancies for longer (similar to300 days) than treatment with low-dose GnRH agonist (similar to200 days). In the majority of heifers and cows treated with GnRH agonist, ovarian follicular growth was restricted to early antral follicles (2-4 mm). The findings indicate that GnRH agonist implants have considerable potential as a practical technology to suppress ovarian activity and control reproduction in female cattle maintained in extensive rangelands environments. The technology also has broader applications in diverse cattle production systems. (C) 2002 Elsevier Science B.V. All rights reserved.

Clarithromycin and prednisolone inhibit cytokine production in chronic rhinosinusitis

Objectives. Long-term, low-dose macrolide therapy is effective in the treatment of chronic rhinosinusitis. The mechanism of its anti-inflammatory effect and how this differs from corticosteroids remains unclear. The effect of clarithromycin and prednisolone on interleukin-5, interleukin-8, and granulocyte-macrophage colony-stimulating factor production by cultured chronic sinusitis nasal mucosa was examined in the study. Study Design and Methods. Nasal mucosa was obtained from 11 patients with chronic sinusitis. This tissue was cultured for 24 hours in the presence of clarithromycin or prednisolone at a variety of concentrations. Cytokine levels were determined by enzyme-linked immunoassay. Results. Clarithromycin and prednisolone each produced significant reductions in interleukin-5, interleukin-8, and granulocyte-macrophage colony-stimulating factor production. There was no significant difference between the effects of clarithromycin and prednisolone. Conclusion: Macrolide antibiotics are capable of inhibiting pro-inflammatory cytokine production in vitro and are as potent as prednisolone. This mechanism is likely to be at least partly responsible for the clinical efficacy of macrolide antibiotics in chronic rhinosinusitis. Key Words. Macrolide, prednisolone, sinusitis, enzyme-linked immunosorbent assay, cytokine.

The therapeutic potential of dopamine modulators on the cardiovascular and renal systems

In the periphery, physiological dopamine increases renal blood flow, decreases renal resistance and acts on the kidney tubule to enhance natriuresis and diuresis. The loss of dopamine function may be involoved in the deterioration in kidney function associated with ageing and may have a role in the pathogenesis of hypertension and diabetes. Intravenous dopamine is used as a positive inotrope in the treatment of acute heart failure and cardiogenic shock and as a diuretic in renal failure. The clinical uses of dopamine are limited, as it must be given intravenously, and also has widespread effects. The levels of peripheral dopamine can be increased by the administration of L-dopa to increase synthesis, prodrugs to release dopamine (docarpamine, glu-dopa) or by inhibiting the breakdown of dopamine (nitecapone). Preliminary clinical trials suggest that docarpamine may be useful in patients with low cardiac output syndrome after cardiac surgery and in refractory cirrhotic ascites. Ibopamine is an agonist at dopamine D1 and D2 receptors, which may retard the progression of chronic renal failure. Gludopa is selective for the kidney thus avoiding widespread side effects. The early clinical studies with ibopamine as a diuretic in heart failure were favourable but the subsequent large mortality study showed that ibopamine increased mortality. Fenoldopam is a selective dopamine D1 receptor agonist. Intravenous fenoldopam may be useful in the treatment of hypertension associated with coronary artery bypass surgery or in hypertensive emergencies. Although our understanding of physiological and pathological roles of peripheral dopamine has been increasing rapidly in recent times, we still need more information to allow the design of clinically useful drugs that modify these roles. One priority is an orally-active selective dopamine D1 receptor agonist.