AWD 140-190: a potent anticonvulsant in the amygdala-kindling model of partial epilepsy

PURPOSE: Evaluation of the effect of the new anticonvulsant drug, AWD 140-190 [4-(p-bromophenyl)-3-morpholino-1H-pyrrole-2-carboxylic acid methyl ester] on focally induced seizures and on epileptogenesis in the kindling model. METHODS: Effects of AWD 140-190 were studied in amygdala kindled rats after oral and intraperitoneal administration. In addition, the effect on kindling development was evaluated. In all experiments, behavioral changes in the rats in response to AWD 140-190 were monitored closely. RESULTS: AWD 140-190 exerted potent anticonvulsant activity against focal seizures. After intraperitoneal and oral administration in fully kindled rats, the substance dose-dependently increased the threshold for induction of afterdischarges starting at 15 mg/kg. AWD 140-190 only weakly influenced the seizure severity of the animals after stimulation at the elevated afterdischarge threshold current. No adverse effects were observed up to 30 mg/kg after intraperitoneal and oral administration in the open field and in the rotarod test. No differences were found between kindled and nonkindled rats when comparing neurotoxicity of AWD 140-190. Prolonged treatment with AWD 140-190 during kindling acquisition did not prevent kindling, but significantly retarded the development of fully kindled seizures during the treatment. CONCLUSIONS: This study demonstrates that AWD 140-190 has anticonvulsant effects in the amygdala kindling model in rats, suggesting that the substance is particularly effective against partial seizures. AWD 140-190 is orally active and devoid of neurotoxic effects in anticonvulsant doses, thus indicating that this compound has potential for antiepileptic therapy. AWD 140-190 retards the kindling development during the treatment. This effect could be explained by the acute anticonvulsant effect of the substance.     

Expression and immunogenicity of oncofetal antigen-immature laminin receptor in human renal cell carcinoma

PURPOSE: The 32 to 44 kDa. oncofetal antigen-immature laminin receptor (OFA-iLR) is a multifunctional protein expressed by various tumors, including breast, lung, ovary and prostate carcinoma as well as lymphoma. OFA-iLR has been implicated in tumor invasiveness, metastasis and growth. Interferon-gamma producing effector T cells and interleukin (IL)-10 producing suppressor T cells specific for OFA-iLR have been described. MATERIALS AND METHODS: The 43515 IgG2a anti-OFA-iLR monoclonal antibody was used to detect OFA-iLR expression in human renal cell carcinoma tissue by flow cytometry and immunoblotting. Spontaneous or therapy induced immune responses against OFA-iLR were determined in patients with metastatic renal cell carcinoma. Proliferative and cytokine (interferon-gamma and IL-10) responses of peripheral blood mononuclear cells from patients with renal cell carcinoma against recombinant OFA-iLR were assessed. RESULTS: Using flow cytometry OFA-iLR was detected in all 13 tumors tested. Immunoblotting revealed differences in OFA-iLR expression in renal cell carcinoma and normal kidney tissue. OFA-iLR specific proliferative and cytokine responses of mononuclear cells were detected in all 6 patients tested. Importantly evidence was also obtained that treating metastatic renal cell carcinoma with tumor lysate pulsed dendritic cells would enhance OFA-iLR specific immunity. CONCLUSIONS: This study demonstrates that OFA-iLR is an immunogenic tumor associated antigen in human renal cell carcinoma. OFA-iLR specific effector T cells producing interferon-gamma may have a role in the control of tumor growth, whereas suppressor T cells producing IL-10 may promote tumor tolerance and, thus, tumor progression.     

Epstein-Barr virus episomes as targets for cigarette smoke- and gamma- irradiation-induced DNA damage: studies on the EBNA-1 region by a new gene-specific technique

Following our demonstration of cytochrome P450-independent DNA damage induced by aqueous solutions of cigarette smoke in human mucosal cells in vivo, and in a lymphoblastoid cell line, we have developed a new technique to demonstrate gene-region specific DNA damage, with the EBNA- 1 gene present in multiple nuclear matrix-attached episomes in Raji cells serving as an amplified target. DNA was extracted from Raji cells treated by gamma-irradiation or aqueous solutions of cigarette smoke; adducted bases or other damage were removed chemically by depurination/alkali treatment. Single-strand breaks induced directly by cigarette smoke as well as DNA cleaved at the site of former adducts were end-labelled either with alpha-[32P]dCTP or with biotin-16-dUTP. With 32P-labelling, a dose-dependent increase in DNA labelling was seen for different concentrations of cigarette smoke; undiluted smoke produced a similar amount of damage as 22.4 Gy of gamma-irradiation. For isolation of DNA regions that contained biotin label at the sites of former damage, DNA was cut by restriction endonucleases and 3-kb- fragments including the target gene, EBV-EBNA-1, were isolated by agarose-gel electrophoresis. Those containing biotin were selected on streptavidin-coated magnetic beads. PCR amplification of the bound DNA revealed EBNA-1 DNA only when cells were pretreated with either cigarette smoke or gamma-irradiation. The presented method thus provides a new approach for detecting gene-specific damage in a readily accessible target, EBV episomes. The method is also potentially applicable for studying single-copy genes such as p53, the types of adducts involved, and quantitative aspects of DNA damage and its repair.      

Immunoaffinity purification and gas chromatography--mass spectrometric quantification of 3-alkyladenines in urine: metabolism studies and basal excretion levels in man

Immunoaffinity gels were prepared by coupling monoclonal antibody(Mab) EM-6–47 to protein A —Sepharose, and wereused to make small columns retaining 3-alkyladenines (3-alkAde)of diverse structure. An analytical procedure for determinationof 3-methyladenine (3-MeAde), 3-ethyladenine (3-EtAde), 3-(2-hydroxyethyl)adenine(3-HOEtAde) and 3-benzyladenine (3-BzAde) was developed. Deuteratedinternal standards (d₃-3-MeAde, d₅-3-EtAde, d₄-3-HOEtAde andd₇-3-BzAde) were synthesized and added to urine samples priorto immunoaffinity purification. 3-alkAde were separated andquantitated as tert-butyl-dimethylsilyl (TBDMS) derivativesby capillary gas chromatography—low resolution mass spectrometry(GC—MS). Detection limits for 3-MeAde, 3-EtAde and 3-HOEtAdewere 0.2 pmol/ml urine and for 3-BzAde, 1 pmol/ml urine. Studiesin two volunteers showed that 3-MeAde and 3-HOEtAde were excretedalmost quantitatively (>90%) within 24 h, that 3-EtAde wasless well excreted (67–74%) and that 3-BzAde was poorlyexcreted (21–25%). Studies on basal levels of 3-alkAdeurinary excretion in three volunteers showed that 3-MeAde was>90% derived from the diet as the preformed product. 3-HOEtAdewas present at     

Increased endogenous N-nitrosamine and nitrate formation by induction of nitric oxide synthase in rats with acute hepatic injury caused by Propionibacterium acnes and lipopolysaccharide administraction

In rats treated i.v. with heat-killed Propionibacterium acnes(100 mg/kg body wt), followed 5 days later by an i.v. dose ofEscherichia coli lipopolysaccharide (LPS, 1 mg/kg body wt),acute hepatic cell necrosis was accompanied by significant inductionof nitric oxide (NO) synthase activity in the liver. Endogenousnitrosation of thiazolidine 4-carboxylic acid (TCA, 50 µmol/rat)administered by three different routes (i.v., i.p. and p.o.)5 h after LPS injection to the P.acnes-treated rats was assessedby analysing its nitrosated product (NTCA) excreted in 24 hurine. The amounts of NTCA formed in vivo after i.v., i.p. andp.o. administration of TCA were 4.07 ± 1.00, 5.79 ±2.15 and 58.3 ± 20.7 nmol/rat (n = 5–10) respectively,which were about 5-, 10- and 8-fold greater than those excretedby rats which had not been treated with P.acnes and LPS butreceived TCA by the same route. Nitrate concentration in plasmaand NO synthase activity in the liver started to increase within2.5 h after LPS injection, reached a maximum at 7.5 h and remainedat high levels for serveral further hours. Levels of nitriteand nitrate in gastric contents were also increased significantlyafter LPS administration. The co-administration of N     

Analysis of radon-associated squamous cell carcinomas of the lung for a p53 gene hotspot mutation

Squamous cell lung carcinomas (SCC) from former employees of the Wismut uranium mining company (Saxony, Germany) were obtained from the Stollberg Archive in order to screen for p53 tumour suppressor gene codon 249 arg-->met hotspot mutations, a putative molecular bio-dosimeter of alpha-particle (radon) exposure (Taylor et al (1994) Lancet 343: 86-87; McDonald et al (1995) Cancer Epidemiol Biomarkers Prevent 4: 791-793). Of the 29 archived samples of SCC meeting quality criteria for DNA analysis by polymerase chain reaction (PCR) and Haelll restriction enzyme digestion, two tumours were found that harboured this mutation. DNA sequencing confirmed the presence of a G to T base substitution within the Haelll site spanning codons 249 and 250 of the p53 gene that results in replacement of arginine (wild-type) by methionine at residue 249. When these data are combined with those from our previous study of tumours from the Stollberg Archive in which 50 lung tumours were examined, (including nine SCCs), we conclude that the G-->T (arg-->met) codon 249 mutation prevalence in the Wismut miner cohort is not sharply elevated in lung cancers in general (two mutations/79 tumours), or specifically in SCCs of the lung (two mutations/38 SCC) when compared to data from lung cancer patients with no reported occupational exposure to radon gas.     

Incidence of urethral tumor involvement in 910 men with bladder cancer

Summary Urethral tumor involvement was examined in 910 patients treated for bladder cancer at a single institution over a period of 25 years. The overall incidence in 2,052 primary and recurrent bladder-tumor events was 6.1%. Risk factors for urethral tumor occurrence are tumors at the bladder neck and recurrent multifocal tumors. Carcinoma in situ (CIS) of the bladder not involving the bladder neck and muscle-invasive tumors with or without lymph-node involvement are not significantly correlated with urethral cancer. Patients at risk for urethral tumors as outlined should be worked up very carefully (multiple urethral biopsies and/or urethral brushings, frozen section of the membranous urethra) before they are considered for an orthotopic neobladder. Altogether, 17 of 89 patients had 1–6 urethral tumor recurrences. The majority of urethral tumors were treated with a single conservative treatment session and did not recur thereafter. A conservative approach toward superficial urethral tumor recurrences in patients with an orthotopic neobladder to the urethra may therefore be feasible.     

Experiments in animals on the pharmacological effects of metipranolol in comparison with propranolol and pindolol.

The beta-blocking agent 1-(4-acetoxy-2,3,5-trimethylphenyloxy)-3-isopropylamino-propan-2-ol (metipranolol) was compared with propranolol and pindolol. The beta-blocking activity on isoproterenol induced tachycardia was determined in rabbits (ED250bpm). The following doses in microgram/kg i.v. were required to produce the same inhibition: 410 propranolol; 160 metipranolol; 130 pindolol. When intrinsic sympathomimetic activity was determined in reserpinized rats metipranolol was found to produce less than 10% of the increase in heart rate induced by a standard isoproterenol dose (1 mg/kg i.p.), whereas propranolol caused less than 20% and pindolol ca. 60%. The membrane stabilising activity, determined by the elevation of the fibrillation threshold (ED delta100muA) in rabbit hearts, was found to be greatest with propranolol (0.98 mg/kg i.v.) and least with metipranolol (1.68 mg/kg), with pindolol occupying an intermediate position (1.10 mg/kg). The cardioprotective action to hypoxia stress of metipranolol in rats was found to be the best, requiring a dose of only 5 microgram/kg i.p. compared with values of 28 microgram/kg for pindolol and 250 microgram/kg for propranolol. These results show that metipranolol has a high beta-sympatholytic activity which is not accompanied by either marked intrinsic activity or membrane-stabilising properties. The relatively marked cardioprotective action, which is probably due to a metabolic action, is particularly noteworthy.