Memory-impairing effects of local anesthetics in an elevated plus-maze test in mice

Detailed deletion mapping of chromosome 6q has shown that the highest percentage of loss of heterozygosity (LOH) is located at 6q25-q27 and suggested that an ovarian cancer associated tumor suppressor gene may reside in this region. To further define the smallest region of common loss, we used 12 tandem repeat markers spanning a region no more than 18 cM, located between 6q25.1 and 6q26, to examine allelic loss in 54 fresh and paraffin embedded invasive ovarian epithelial tumor tissues. Loss of heterozygosity was observed more frequently at the loci defined by marker D6S473 (14 of 32 informative cases, 44%) and marker D6S448 (17 of 40 informative cases, 43%). Detailed mapping of chromosome 6q25-q26 in these tumor samples identified a 4 cM minimal region of LOH between markers D6S473 and D6S448 (6q25.1-q25.2). Loss of heterozygosity at D6S473 correlated significantly both with serous versus non-serous ovarian tumors (P=0.040) and with high grade versus low grade specimens (P=0.023). The results suggest that a 4 cM deletion unit located at 6q25.1-q25.2 may contain the putative tumor suppressor gene which may play a role in the development and progression of human invasive epithelial ovarian carcinomas (IEOC).     

Improvement by FUB 181, a novel histamine H3-receptor antagonist, of learning and memory in the elevated plus-maze test in mice

PURPOSE: Initial experimental results of contrast-enhanced pulmonary MR angiography using the new superparamagnetic iron oxide blood pool agent FeO-BPA. METHOD: Pulmonary MRA was performed in 7 domestic pigs using a cardiac-triggered coronal T1-weighted FFE-Sequence before and up to 90 minutes after contrast injection obtained on a 1.5 T magnet with a standard gradient equipment. A dose of 4 mg Fe/kg bodyweight was used for MRA. The images were qualitatively assessed and compared with X-ray i.v.-DSA. RESULTS: The injection of FeO-BPA allows the acquisition of unsaturated in-plane images of the pulmonary vascular tree down to the first order subsegmental branches including vessel diameters of approximately 1.5 mm. In the normal non-occluded vasculature, no signal void is seen in the TE range of 2.8-5.5 ms secondary to exceeding susceptibility effects which are caused by the iron oxide accumulation. Even 90 minutes after injection of FeO-BPA, assessment of the pulmonary vasculature is still satisfactory. CONCLUSIONS: In the animal experiment, the use of the blood pool agent FeO-BPA provides detailed pulmonary angiograms even on a magnet with a conventional gradient system. The major advantage is the comfortable diagnostic window of > 1.5 hours which also portends its utility for future MR-guided pulmonary interventions.     

Ethological analysis of cholecystokinin (CCKA and CCKB) receptor ligands in the elevated plus-maze test of anxiety in mice

Membranes for determination of maltose, lactose and sucrose with the use of an oxygen electrode are described. They were obtained by immobilization of glucose oxidase with suitable disaccharide hydrolase in gelatin or albumin. For the sucrose determination mutarotase was also used. The membranes contained one, two or three enzymes working in sequence. The enzyme composition of the membranes influenced the linear range slightly and the value of electrode response considerably. The electrode response of the maltose membranes was also affected by the immobilization material. The measurements with the use of enzymatic membranes were very sensitive to temperature and, to some degree, the pH of the sample. Stability of the membranes was differentiated. The albumin membranes for sucrose determination generated almost the same signal after 1 month of frequent use, whereas the gelatin membranes for lactose determination lost about 85% of the initial activity after 1 week of operation. The electrode response of both the gelatin and albumin maltose membranes decreased in the same time by about 50%.     

Anxiogenic effect of phenylethylamine and amphetamine in the elevated plus-maze in mice and its attenuation by ethanol

In previous experiments beta-phenylethylamine (PEA), like the standard anxiogens caffeine, pentylenetetrazole, and yohimbine, has exhibited an anxiogenic effect in the two animal models of anxiety: the social interaction test and the conflict situation test. In the present study, PEA acts as an anxiogen in an elevated plus-maze, diminishing (compared to controls) the ratio of entries into open arms over the total number of entries and shortening the time spent in the open arms. DL-Amphetamine sulfate (AMPH) also had a similar action. These data support the previous suggestion that PEA may belong to the group of endogenous anxiety-inducing compounds. Pretreatment with ethanol prevented the effects of both PEA and AMPH.     

Induction of B-cell lymphoma in BALB/c nude mice with an ecotropic, B-tropic helper virus present in the murine AIDS virus stock

The pathogenicities of the murine AIDS (MAIDS) virus complex (LP-BM5) and ecotropic helper virus (BM5eco) isolated from the complex to BALB/c nude mice were studied to elucidate the possible role of replication-competent helper virus in inducing the monoclonal outgrowth of lymphoid cells. Neither LP-BM5 nor BM5eco was pathogenic in adult BALB/c nude mice. However, B-cell lymphoma developed with a very high frequency when either virus was inoculated into newborn BALB/c nude (nu/nu) mice. The cells from the B-cell lymphoma were easily transplanted into nude mice. These results suggested that ecotropic helper virus in the MAIDS virus complex plays an important role in inducing the monoclonal outgrowth of lymphoid cells under immunodeficient conditions caused by defective virus.     

ACEA-1328, an NMDA receptor antagonist, increases the potency of morphine and U50,488H in the tail flick test in mice

The effect of 5-nitro-6,7-dimethyl-1,4-dihydro-2,3-quinoxalinedione (ACEA-1328), a competitive and systemically bioavailable NMDA receptor/glycine site antagonist, was examined on opioid-induced antinociception in the tail flick test. Swiss Webster mice were injected with ACEA-1328 either alone or in combination with morphine or (+/-)-trans-U-50488 methanesulfonate (U50,488H), a mu- and a kappa-opioid receptor agonist, respectively, and tested for antinociception. Systemic administration of ACEA-1328 alone increased the tail flick latencies with an ED50 of approximately 45 mg kg-1. Concurrent administration of ACEA-1328 with morphine, or U50,488H, at doses that did not affect tail flick latencies, potentiated the antinociceptive effect of the opioid analgesics and vice versa. Naloxone, an opioid receptor antagonist, while not modifying the effect of ACEA-1328, did block the augmentation, suggesting that opioid receptors might be involved in the latter effect. 5-Aza-7-chloro-4-hydroxy-3-(m-phenoxyphenyl)quinoline-2(1H)-one (ACEA-0762), a selective NMDA receptor/glycine site antagonist, also showed enhancement of the antinociceptive effect of morphine and U50,488H. However, concurrent administration of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzol[f]quinoxaline (NBQX), a selective non-NMDA receptor antagonist, with morphine did not alter the antinociceptive potency of the opioid analgesic. Overall, the data suggest that ACEA-1328 may increase the potency of the opioid analgesics by antagonising the glycine site associated with the NMDA receptor.     

Prevention of onset in an insulin-dependent diabetes mellitus model, NOD mice, by oral feeding of Lactobacillus casei

Allogeneic bone marrow transplantation is the only currently available curative treatment for myelodysplastic syndromes (MDSs) but can be used only in the minority of patients (10%) who are younger than 55 years or so and for whom an HLA-identical donor is available. Each year in Europe, about 100 patients with MDSs receive an autologous bone marrow transplant. This procedure is usually indicated as first-line treatment, except in patients without excess of blasts or complex cytogenetic abnormalities. In forms with excess of blasts, chemotherapy prior to bone marrow transplantation deserves discussion. Autologous bone marrow transplants or the more recent technique involving transplantation of autologous peripheral stem cells can be considered in patients who have achieved a complete remission under aggressive chemotherapy. This method has been followed by higher recurrence rates in patients with MDSs than in those with de novo acute myeloblastic leukemia, and randomized studies are under way to compare it with aggressive maintenance chemotherapy.     

Restoration of an early, progressive defect in responsiveness to T-cell activation in lupus mice by exogenous IL-2

Splenic T-cells from lupus strain (NZB/W F1, Mrl/lpr) mice lack the ability to respond to concanavalin A (Con A) by secretion of IL-2 and hence expression of IL-2 receptor and proliferation. These defects were found not only in an aged group (> 5 months) of mice in which obvious clinical 'SLE like' symptoms and elevated levels of serum autoantibodies were observed, but also in mice as young as 4-wk. We demonstrate here that the defective mitogenic activation of T-cells from lupus mice is due to the inability of T-helper cells to produce IL-2 and this defect can be restored by exogenous IL-2 in vitro. Con A-induced cell proliferation and IL-2 receptor expression on CD3+ cells from lupus mice occur only in the presence of exogenous IL-2, whereas normal T-cells from BALB/c and CBA control mice are activated by the mitogen and undergo complete cell cycling in the absence of exogenous IL-2, as they are able to secrete sufficient endogenous IL-2. The detection of impaired T-helper function in young lupus mice, before development of overt disease, and the reversible nature of the defect indicate that defective IL-2 activity may be fundamental to the mechanism of development of pathology in SLE.     

Intraepithelial infiltration by mast cells with both connective tissue-type and mucosal-type characteristics in gut, trachea, and kidneys of IL-9 transgenic mice

IL-9 transgenic mice were analyzed for the presence of mast cells in different tissues. In these mice, increased mast cell infiltration was found in the gastric and intestinal epithelium as well as in the upper airways and kidney epithelium, but not in other organs, such as skin. IL-9 transgenic mast cells do not show signs of massive degranulation such as that found in IL-4 transgenic mice and are not involved in spontaneous pathologic changes. Gastric mast cells showed a phenotype related to connective-type mast cells, since they were stained by safranin, and strong expression of mouse mast cell protease-4 and -5 was found in this organ. However, they also expressed proteases related to the mucosal cell type, such as mouse mast cell protease-1 and -2. In vitro, although IL-9 by itself did not induce mast cell development from bone marrow progenitors, it strongly synergized with stem cell factor for the growth and differentiation of mast cells expressing the same protease pattern as that observed in IL-9 transgenic mice. Since constitutive stem cell factor expression was observed in vivo, and anti-c-Kit Abs inhibited IL-9 transgenic mastocytosis in the gut, this synergistic combination of factors is likely to be responsible for the mastocytosis observed in IL-9 transgenic mice. Taken together, these data demonstrate that IL-9 induces the in vivo amplification of a nonclassical mast cell subset with a mucosal localization but expressing proteases characteristic of both connective tissue-type and mucosal mast cells.     

Activation of the metaphase checkpoint and an apoptosis programme in the early zebrafish embryo, by treatment with the spindle-destabilising agent nocodazole

We have studied the developmental activation of the metaphase checkpoint, and the consequences of activating this checkpoint, in the zebrafish embryo. (1) Treatment with nocodazole (a microtubule destabiliser) before mid-blastula transition (MBT) produces complete destruction of all nuclei in the deep cell layer of the embryo. In contrast, nocodazole treatment after MBT efficiently produces metaphase arrest in this cell layer. Thus, the metaphase checkpoint becomes activated at MBT. (2) Although a metaphase arrest is induced by nocodazole, it is not induced by paclitaxel (a microtubule stabiliser). Thus the metaphase checkpoint appears to sense a destabilisation, but not a stabilisation, of spindle microtubules. (3) Metaphase-arrested cells (in nocodazole) can be driven into the next interphase by adding the Ca2+-specific ionophore A23187. Thus, a Ca2+-signalling pathway lies downstream of, or parallel to, the metaphase checkpoint. (4) After mid-gastrula stage, treatment with nocodazole produces DNA fragmentation in all three cell layers. In the enveloping epithelial monolayer (EVL), this is associated with a classical apoptotic phenotype. In the deep layer, it is associated with an unusual, highly condensed nuclear state that is entered directly from metaphase arrest. Thus, after the mid-gastrula stage, the embryo responds to nocodazle by undergoing apoptosis. (5) Nocodazole-induced apoptosis in the deep cell layer can be blocked by the caspase-1,4,5 inhibitors Ac-YVAD-CHO and Ac-YVAD-CMK. This suggests that a homologue of the C. elegans ced-9-ced-4-ced-3 pathway is involved in control over apoptosis in the early zebrafish embryo.     

Inhibition of low density lipoprotein oxidation in vitro by the 6- and 7-hydroxy-metabolites of doxazosin, an alpha 1-adrenergic antihypertensive agent

Heart rate and flow velocity in the common carotid artery were examined on eight subjects in various underwater conditions. The heart rate decreased more markedly during whole-body immersion than during head-out immersion. The end-diastolic flow velocity and the flow-velocity integral in a cardiac cycle during whole-body immersion increased more markedly than during head-out immersion. Differences in heart rate and flow velocity were not detected between sitting and prone positions in whole-body immersion. Heart rate increased at the beginning and after underwater swimming. The peak systolic flow velocity increased significantly after the end of underwater swimming. The flow-velocity integral in a cardiac cycle decreased during underwater swimming, but in a minute did not change significantly. These results suggest that facial immersion is important in eliciting pronounced carotid artery flow-velocity response in addition to the heart-rate response and that underwater exercise influences these responses. However, underwater posture apparently does not influence them.     

A single cell gel electrophoresis technique for the detection of DNA damage induced by ACNU, an alkylating agent or irradiation in murine glioma cell lines

A simple and convenient technique for in situ quantification of DNA damage induced by 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloro-ethyl)-3-nitrosourea hydrochloride (ACNU) an alkylating agent, or irradiation was demonstrated in C6 glioma cells using a single cell gel electrophoresis. Treatment with ACNU or irradiation caused a dose dependent DNA damage which was detected by measuring the length of migration of fragmentary DNA in individual cells. Wild type C6 cells treated with ACNU (0, 10, 30, 60 micrograms ml-1) for one hour showed longer distance of migration of DNA than the ACNU-resistant subtype cells (C6R), indicating that ACNU-sensitive C6 cells were more vulnerable to ACNU than C6R cells. The results of DNA migration in C6 and C6R cells treated with ACNU were consistent with that from MTT assay which had been regarded as a standard method for chemosensitivity test. Furthermore, a time course study for DNA repair activity of C6 and C6R cells was also performed by measuring the length of DNA migration after incubation (0, 15, 30, 60, 120 min) of cells treated with 60 micrograms ml-1 ACNU. C6R cells repaired DNA damage more rapidly than C6 cells. In addition, the technique was also used to measure the DNA damage in C6 cells exposed to 0, 2, 6, 8, 10 Gy of x-ray irradiation, and a dose-dependent DNA migration after radiation injury was observed. This technique appears to be simple and useful for assessing chemosensitivity or radiosensitivity in individual glioma cells.     

Pharmacokinetics, oral bioavailability, and metabolism in mice and cynomolgus monkeys of (2'R,5'S-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine, an agent active against human immunodeficiency virus and human hepatitis B virus

(2'R,5'S-)-cis-5-Fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine (524W91) is a nucleoside analog with potent anti-human immunodeficiency virus and anti-human hepatitis B virus activities in vitro. The pharmacokinetics and bioavailability of 524W91 after oral dosing were studied in mice dosed with 10, 100, and 600 mg of 524W91 per kg of body weight by the oral and intravenous routes. Cynomolgus monkeys were dosed with 10 and 80 mg of 524W91 per kg. In both species, the clearance of 524W91 was rapid, via the kidney, and was independent of dose. In monkeys, the total body clearance of 10 mg of 524W91 per kg was 0.7 +/- 0.1 liter/h/kg, and the volume of distribution at steady state was 0.8 +/- 0.02 liter/kg. The terminal elimination half-life was 1.0 +/- 0.2 h. The absolute bioavailability after oral dosing was 63% +/- 4% at 10 mg/kg. Concentrations of 524W91 in the cerebrospinal fluid were 4% +/- 0.7% of the corresponding levels in plasma. In mice, the total clearance of 10 mg of 524W91 per kg was 2.3 liters/kg/h, and the volume of distribution at steady state was 0.9 liter/kg. Absolute bioavailability in mice after oral dosing was 96% at a dose of 10 mg/kg. The metabolism of orally administered [6-3H]524W91 was studied in cynomolgus monkeys at a dose of 80 mg/kg and in mice at a dose of 120 mg/kg. Monkeys excreted 41% +/- 6% of the radioactive dose in the 0- to 72-h urine, 33% +/- 10% in the feces, and 10% +/- 7% in the cage wash. Unchanged 524W91 was 64% of the total radiolabeled drug recovered in the urine. The glucuronide was a minor urinary metabolite. 5-Fluorouracil was not detected (less than 0.02% of the dose). Mice dosed orally with 120 mg of [6-3H]524W91 per kg excreted 67% +/- 7% of the radiolable in the )- to 48-h urine. Small amounts of the 3' -sulfoxide and glucuronide metabolites were observed in the urine, but 5-fluorouracil was not detected. Good bioavailability after oral dosing and resistance to metabolism recommend 524W91 for further preclinical evaluation.