Netrin-1 protects hypoxia-induced mitochondrial apoptosis through HSP27 expression via DCC- and integrin α6ß4-dependent Akt, GSK-3ß, and HSF-1 in mesenchymal stem cells.

Netrin (Ntn) has the potential to be successfully applied as an anti-apoptotic agent with a high affinity for tissue, for therapeutic strategies of umbilical cord blood-derived mesenchymal stem cells (UCB-MSC), although the mechanism by which Ntn-1 protects hypoxic injury has yet to be identified. Therefore, the present study examined the effect of Ntn-1 on hypoxia-induced UCB-MSC apoptosis, as well as the potential underlying mechanisms of its protective effect. Hypoxia (72 h) reduced cell viability (MTT reduction, and [(3)H]-thymidine incorporation) and cell number, and induced apoptosis (annexin and/or PI positive), which were reversed by Ntn-1 (10 ng/ml). Moreover, Ntn-1 decreased the increase of hypoxia-induced Bax, cleaved caspase-9, and -3, but blocked the decrease of hypoxia-reduced Bcl-2. Next, in order to examine the Ntn-1-related signaling cascade in the protection of hypoxic injury, we analyzed six Ntn receptors in UCB-MSC. We identified deleted in colorectal cancer (DCC) and integrin (IN) α6ß4, except uncoordinated family member (UNC) 5A-C, and neogenin. Among them, IN α6ß4 only was detected in lipid raft fractions. In addition, Ntn-1 induced the dissociation of DCC and APPL-1 complex, thereby stimulating the formation of APPL-1 and Akt2 complex. Ntn-1 also reversed the hypoxia-induced decrease of Akt and glycogen synthase kinase 3ß (GSK-3ß) phosphorylation, which is involved in heat shock factor-1 (HSF-1) expression. Ntn-1-induced phospho-Akt and -GSK-3ß were inhibited by DCC function-blocking antibody, IN a6b4 function-blocking antibody, and the Akt inhibitor. Hypoxia and/or Ntn-1 stimulated heat shock protein (HSP)27 expression, which was blocked by HSF-1-specific small interfering RNA (siRNA). Furthermore, HSP27-specific siRNA reversed the Ntn-1-induced increase of phospho-Akt. Additionally, HSP27-specific siRNA attenuated the Ntn-1-reduced loss of mitochondrial membrane injury via the inhibition of cytochrome c (cyt c) release and formation of cyt c and HSP27 complex. Moreover, the inhibition of each signaling protein attenuated Ntn-1-induced blockage of apoptosis. In conclusion, Ntn-1-induced HSP27 protected hypoxic injury-related UCB-MSC apoptosis through DCC- and IN α6ß4-dependent Akt, GSK-3ß, and HSF-1 signaling pathways.

In vitro bioactivity and osteoblast response of porous NiTi synthesized by SHS using nanocrystalline Ni-Ti reaction agent.

Porous NiTi with an average porosity of 55 vol % and a general pore size of 100-600 microm was synthesized by self-propagating high temperature synthesis (SHS) with the addition of mechanically alloyed nanocrystalline Ni-Ti as the reaction agent. The SHS of porous NiTi using elemental powders was also performed for comparison. To enhance the bioactivity of the metal surface, porous NiTi synthesized by nanocrystalline Ni-Ti was subjected to chemical treatment to form a layer of TiO(2) coating. The porous NiTi with TiO(2) coating was subsequently immersed in a simulated body fluid (SBF) to investigate its apatite forming ability. The effects of the addition of nanocrystalline Ni-Ti as reaction agent and the application of apatite coating on osteoblastic behavior were studied in primary cultures of human osteoblast cells. Results showed that the main phases in porous NiTi synthesized by elemental powders were NiTi, Ti(2)Ni, and unreacted free Ni. By using nanocrystalline Ni-Ti as reaction agent, the secondary intermetallic phase of Ti(2)Ni was significantly reduced and the free Ni was eliminated. TiO(2) coating with anatase phase was formed on the surface of porous NiTi after the chemical treatment. A layer consisting of nanocrystalline carbonate-containing apatite was formed on the surface of TiO(2) coating after soaking in SBF. The preliminary cell culture studies showed that the porous NiTi synthesized with the addition of nanocrystalline Ni-Ti attracted marked attachment and proliferation of the osteoblast cells. This gives the evidence of the potential biomedical applications of the porous NiTi.

Biomimetic deposition of apatite coating on surface-modified NiTi alloy.

TiO(2) coatings were prepared on NiTi alloy by heat treatment in air at 300, 400, 600 and 800 degrees C. The heat-treated NiTi alloy was subsequently immersed in a simulated body fluid for the biomimetic deposition of the apatite layer onto the surface of TiO(2) coating. The apatite coatings as well as the surface oxide layer on NiTi alloy were characterized using scanning electron microscopy equipped with energy dispersive spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy and Raman spectroscopy. Results showed the samples heat-treated at 600 degrees C produced a layer of anatase and rutile TiO(2) on the surface of NiTi. No TiO(2) was detected on the surface of NiTi after heat treatment at 300 and 400 degrees C by X-ray diffraction, while rutile was formed on the surface of the 800 degrees C heat-treated sample. It was found that the 600 degrees C heat-treated NiTi induced a layer consisted of microcrystalline carbonate containing hydroxyapatite on its surface most effectively, while 300 and 400 degrees C heat-treated NiTi did not form apatite. This was due to the presence of anatase and/or rutile in the 600 and 800 degrees C heat-treated NiTi which could provide atomic arrangements in their crystal structures suitable for the epitaxy of apatite crystals, and anatase had better apatite-forming ability than rutile. XPS and Raman results revealed that this apatite layer was a carbonated and non-stoichiometric apatite with Ca/P ratio of 1.53, which was similar to the human bone. The formation of apatite on 600 degrees C heat-treated NiTi following immersion in SBF for 3 days indicated that the surface modified NiTi possessed excellent bioactivity.

Depletion of penicillin G residues in tissues, plasma and injection sites of market pigs injected intramuscularly with procaine penicillin G.

Procaine penicillin G was administered by intramuscular (i.m.) injection to groups of healthy 100 kg market pigs at the approved label dose (15,000 IU/kg body weight), once daily for three consecutive days; or an extra-label dose (66,000 IU/kg body weight), once daily for five consecutive days. Penicillin G residue depletion was followed in plasma, tissue and injection sites using a liquid chromatographic method. Groups of pigs were killed 1, 2, 3, 4, 5 and 8 days after the last injection with the label dose. Penicillin G was not detected in liver after 1 day of withdrawal, in muscle and fat after 2 days of withdrawal, in plasma after 4 days of withdrawal, in skin after 5 days of withdrawal, or in kidney and the injection sites after 8 days of withdrawal. Other groups of pigs were killed 1, 2, 3, 5 and 7 days after injection with the extra-label dose. In these pigs penicillin G was not found in liver after 2 days of withdrawal, in fat after 3 days of withdrawal, or in the muscle, skin, plasma and injection sites after 7 days of withdrawal. Penicillin G was found at all times in the kidneys of the groups of pigs that received the high dose. The technique used for neck injections was critical to obtain intramuscular rather than intermuscular injections. The Bureau of Veterinary Drugs, Health Protection Branch, Health Canada calculated that the appropriate withdrawal period for pigs was 8 days for a dose of 15,000 IU procaine penicillin G/kg body weight and 15 days for a dose of 66,000 IU/kg.

Dietary intake estimates as a means to the harmonization of maximum residue levels for veterinary drugs. II. Proposed application to the Free Trade Agreement between the United States and Canada.

The Free Trade Agreement between the United States and Canada (FTA) went into effect January 1, 1989. To implement certain provisions of the agreement on technical regulations and standards, the United States Food and Drug Administration's Center for Veterinary Medicine, Health and Welfare Canada's Bureau of Veterinary Drugs, and Agriculture Canada established the Working Group on Veterinary Drug Tolerances. The progress of the working group and its continuing efforts to harmonize tolerances for approximately 15 veterinary drugs are discussed. This paper proposes use of the toxicologically determined acceptable daily intake (ADI) for the drug as the safety standard for reaching conclusions on the acceptability of residues in meat for human consumption. Specifically, the "equivalence" of different MRLs for the same veterinary drug would be determined by considering whether they are likely to result in dietary residues that exceed the other country's ADI for the drug. Estimates are made for the veterinary drugs lasalocid, fenbendazole, morantel tartrate, halofuginone, and tilmicosin. Based on these estimates, the U.S. and Canadian MRLs for each drug would be considered equivalent for trade purposes.

Depletion of penicillin G residues in tissues and injection sites of yearling beef steers dosed with benzathine penicillin G alone or in combination with procaine penicillin G.

The contribution of benzathine penicillin G to residues in tissues and injection sites of yearling beef steers was assessed by treating seven groups of five to seven steers with either benzathine and procaine penicillin G together or benzathine penicillin G alone. Steers were injected with a commercial combination of benzathine and procaine penicillin G according to the Canadian (intramuscular) or United States (subcutaneous) label dosages of 8600 and 8800 IU penicillin G/kg body weight, respectively. They were killed 14 or 30 days after the intramuscular injections, and 30 days after the subcutaneous injections. At the label withdrawal times, Canadian 14 days and United States 30 days, the levels in the injection sites for all of the treatments were 30-60 times above the Canadian and United States' Maximum Residue Limit of 50 micrograms/kg, while liver, kidney and gluteal muscle levels were below the Maximum Residue Limit. Other steers were injected intramuscularly with 24,000 IU benzathine/procaine penicillin G/kg body weight and slaughtered 8, 14 or 50 days after injection. Fifty-day injection site residues were 24 times the Maximum Residue Limit. Another group of steers was injected intramuscularly with benzathine penicillin G alone at 12,000 IU/kg body weight and slaughtered 14 days later. Penicillin G levels in the injection sites were 156 times the Maximum Residue Limit. The persistence of penicillin G residues at the injection sites in all the treatment groups appears to be attributable primarily to benzathine penicillin G. Visual inspection of muscle surfaces did not reliably reveal all injection site lesions in the underlying musculature.

Depletion of intramuscularly and subcutaneously injected procaine penicillin G from tissues and plasma of yearling beef steers.

Withdrawal periods required when doses of 24,000 IU and 66,000 IU of procaine penicillin G/kg body weight were administered to yearling beef steers by intramuscular injection daily for five consecutive days were investigated. These dosages are in excess of product label recommendations, but are in the range of procaine penicillin G dosages that have been administered for the treatment of some feedlot bacterial diseases. The approved dose in Canada is 7,500 IU/kg body weight intramuscularly, once daily, with a withdrawal period of five days. Based on the tissue residue data from this study, the appropriate withdrawal period is ten days for the 24,000 IU/kg body weight dose and 21 days for the 66,000 IU/kg body weight dose when administered intramuscularly to yearling beef steers. In a related study, 18 yearling beef steers received 66,000 IU of procaine penicillin G/kg body weight administered by subcutaneous injection, an extra-label treatment in terms of both dose and route of administration, typical of current practice in some circumstances. Deposits of the drug were visible at subcutaneous injection sites up to ten days after injection, with more inflammation and hemorrhage observed than for intramuscular injections of the same dose. These results suggest that procaine penicillin G should not be administered subcutaneously at high doses; and therefore a withdrawal period was not established for subcutaneous injection.

The action of agonists and antagonists at the histamine H1 receptor and receptor protection studies in guinea pig ileum.

We have examined the ability of histamine and the competitive reversible antihistamines to protect the histamine H1 receptor against alkylation with the 2-haloalkylamines, phenoxybenzamine and SY-14. In isolated guinea pig ileum these irreversible antagonists produce a parallel shift in the dose-response curve to histamine with retention of the maximum response if they are used at concentrations less than about 10(-6)M. Treatment with these 2-haloalkylamines in the presence of a high concentration of histamine did not alter the blocking activity. Thus histamine appears to be unable to protect its own receptor against irreversible blockade. The competitive reversible antagonists, on the other hand, did provide effective protection against irreversible blockade. It is likely that the competitive reversible H1 receptor antagonists have at least some part of their attachment site in common with irreversible antagonists of the 2-haloalkylamine type, while the inability of histamine to provide self-protection suggests that its primary attachment site is different from that of the antagonists.

Inhibition of the cardiac inotropic effect of phenylephrine by a tetramine disulfide with irreversible alpha-adrenoceptor blocking activity.

The newly synthesized alpha-adrenoceptor blocking drug BHC (N,N'-bis[6-(10-methoxybenzyl-amino)-a-hexyl]cystamine) was found to block irreversibly the positive inotropic effect of the sympathomimetic drug phenylephrine on the isolated rat left atrium. BHC was used to test the adrenoceptor interconversion hypothesis which claims that low temperature converts inotropic beta-adrenoceptors in rat atrium and frog ventricle to alpha-adrenoceptors. There was no evidence of adrenoceptor 'interconversion.' In the rat atrium low temperature did not increase the BHC antagonism of phenylephrine and did not cause BHC to inhibit the inotropic effect of noradrenaline or isoprenaline. In the perfused frog heart low temperature did not lead BHC to inhibit the inotropic effect of phenylephrine, adrenaline, or isoprenaline.

Cardiac muscarinic blocking and atropinic blocking effects of a tetramine disulfide and alpha-adrenoceptor blocking activity.

Since many alpha-adrenoceptor blocking drugs were known to have muscarinic blocking activity, BHC, a newly synthesized tetramine disulfide with alpha-adrenoceptor blocking activity, and two of its analogs were tested for muscarinic blocking activity in the isolated guinea pig atrium and ileum. BHC had muscarinic blocking activity. Its potency was higher in the atrium than in the ileum. The cardiac muscarinic blocking potency of the analog that contained carbon atoms instead of sulfur atoms was similar to that of BHC. Lower concentrations of the drugs antagonized inotropic effects of acetylcholine competitively, but higher concentrations had a smaller effect than expected for competitive antagonism. In the atrium BHC competitively inhibited the muscarinic blocking effect of atropine. Its atropinic blocking potency was similar to the muscarinic blocking potency. Quinidine and gallamine also had muscarinic blocking and atropinic blocking effects in the atrium but were about 10 times less potent than BHC.

Stability and biological activity of catecholamines and 5-hydroxytryptamine immobilized to sepharose and glass beads.

[14C]Noradrenaline, [14C]adrenaline, and 5-[14C]hydroxytryptamine were complexed to several types of Sepharose derivatives through an amide or a diazo linkage. Complexes were also made with arylamine glass beads through a diazo linkage. After the reaction the complexes were extensively washed with distilled water or dilute HCl or both, plus other solvents. Radioactivity appeared in the storage solutions and the amount increased with time. When initial low levels of release were achieved by extensive washing, the addition of a different solvent produced a marked increase in release. The biological activity of these complexes was tested on isolated rabbit aortic strips and rat fundus strips. All complexes caused the tissues to contract. The biological activity, however, closely paralleled the release of the ligands from the complexes and the release of ligand was demonstrated through the use of oil immersion and dialysis sac. These data do not support the recent claims that catecholamines bound to glass beads exert their pharmacological effects as covalently bound complexes on isolated tissue. The lack of stability of these complexes indicates the need for caution in drawing conclusions as to the site or the mechanism of action of immobilized catecholamines and other drugs.

The effect of cocaine, reserpine, and 6-hydroxydopamine on the response of the perfused central artery of the rabbit ear to sympathomimetic amines.

The perfused central artery of the rabbit ear was less sensitive to extraluminal than to intraluminal noradrenaline, but the reverse was true for metaraminol, methoxamine, metanephrine, and isoproterenol. No difference was noted between the extraluminal and intraluminal potency of phenylephrine. Cocaine potentiated the effect of extraluminal and intraluminal noradrenaline, but decreased that of intraluminal phenylephrine. Irrespective of the route of administration, the constrictor potencies of the sympathomimetic amines were not affected by cocaine. Arteries of reserpine-treated rabbits were supersensitive to extraluminally and intraluminally applied noradrenaline and phenylephrine, but they were not supersensitive to metaraminol. 6-Hydroxydopamine effectively destroyed adrenergic nerve endings of the central ear artery and increased its responses to both extraluminal and intraluminal noradrenaline and phenylephrine. However, only the constrictor potencies of intraluminally applied metaraminol and methoxamine were enhanced by 6-hydroxydopamine. The apparent discrepancies between the results obtained by various procedures that eliminate or impair the nerve uptake process suggest that the difference in the constrictor potency of extraluminal and intraluminal sympathomimetic amines is probably unrelated to their uptake by nerves located in the adventitio-medial junction of the artery.

Stability of catecholamines and propranolol covalently bound to sepharose and glass beads.

Catecholamines and propranolol were chemically bound to Sepharose and glass beads and washed extensively. The complexes were unstable and bound ligands were gradually released into the supernatants.