Bombesin antagonist prevents CO2 laser-induced promotion of oral cancer.

We previously reported that CO2 laser incisions in carcinogen-initiated fields promoted cancer development and caused release of growth factors. Here we examined the quantitative and additive properties of this tumor-promoting event and examined whether this promotion could be nullified by treatment with a bombesin antagonist, which down-regulates epidermal growth factor receptors. The model used for cancer promotion was the hamster buccal cheek pouch that had been treated with a carcinogen (9,10-dimethyl-1,2-benzanthracene) for 6 weeks, producing premalignant lesions. These lesions would evolve into a cancer eventually without further treatment. Promotion was measured both by increased fluorescence in response to systemically administered Photofrin, measured noninvasively using an in vivo fluorescence photometer, and by the timing of appearance of clinical tumors. Laser incisions (0-3) were made into the hamster cheek 1 week apart, or three incisions were done 1 day apart. Another group of animals received bombesin antagonist RC-3095 for 4 weeks during the time incisions were made, again measuring promotion. Laser incisions 1 week apart produced additive promotion, whereas three incisions 1 day apart were not statistically different from the group receiving only one incision. RC-3095 treatment completely eliminated the promoting effects of incision and totally stopped promotion for the 4-week period of treatment. After discontinuing treatment with RC-3095, lesion progression resumed at the untreated control rate. This work confirms that the promoting event of a laser incision follows a comparable time course to release of growth factors after such an incision and that it can be eliminated by treatment with bombesin antagonists.

Combined transgenic expression of α-galactosidase and α1,2-fucosyltransferase leads to optimal reduction in the major xenoepitope Galα(1,3)Gal

Hyperacute rejection of pig organs by humans involves the interaction of Galα(1,3)Gal with antibodies and complement. Strategies to reduce the amount of xenoantigen Galα(1,3)Gal were investigated by overexpression of human lysosomal α-galactosidase in cultured porcine cells and transgenic mice. The overexpression of human α-galactosidase in cultured porcine endothelial cells and COS cells resulted in a 30-fold reduction of cell surface Galα(1,3)Gal and a 10-fold reduction in cell reactivity with natural human antibodies. Splenocytes from transgenic mice overexpressing human α-galactosidase showed only a 15–25% reduction in binding to natural human anti-Galα(1,3)Gal antibodies; however, this decrease was functionally significant as demonstrated by reduced susceptibility to human antibody-mediated lysis. However, because there is residual Galα(1,3)Gal and degalactosylation results in the exposure of N-acetyllactosamine residues and potential new xenoepitopes, using α-galactosidase alone is unlikely to overcome hyperacute rejection. We previously reported that mice overexpressing human α1,2-fucosyltransferase as a transgene had ≈90% reduced Galα(1,3)Gal levels due to masking of the xenoantigen by fucosylation; we evaluated the effect of overexpressing α-galactosidase and α1,2-fucosyltransferase on Galα(1,3)Gal levels. Galα(1,3)Gal-positive COS cells expressing α1,3-galactosyltransferase, α1,2-fucosyltransferase, and α-galactosidase showed negligible cell surface staining and were not susceptible to lysis by human serum containing antibody and complement. Thus, α1,2-fucosyltransferase and α-galactosidase effectively reduced the expression of Galα(1,3)Gal on the cell surface and could be used to produce transgenic pigs with negligible levels of cell surface Galα(1,3)Gal, thereby having no reactivity with human serum and improving graft survival.

Bax ablation prevents dopaminergic neurodegeneration in the 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damages dopaminergic neurons in the substantia nigra pars compacta (SNpc) as seen in Parkinson's disease. Here, we show that the pro-apoptotic protein Bax is highly expressed in the SNpc and that its ablation attenuates SNpc developmental neuronal apoptosis. In adult mice, there is an up-regulation of Bax in the SNpc after MPTP administration and a decrease in Bcl-2. These changes parallel MPTP-induced dopaminergic neurodegeneration. We also show that mutant mice lacking Bax are significantly more resistant to MPTP than their wild-type littermates. This study demonstrates that Bax plays a critical role in the MPTP neurotoxic process and suggests that targeting Bax may provide protective benefit in the treatment of Parkinson's disease.

Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

The reaction of the old yellow enzyme and reduced flavins with organic nitrate esters has been studied. Reduced flavins have been found to react readily with glycerin trinitrate (GTN ) (nitroglycerin) and propylene dinitrate, with rate constants at pH 7.0, 25°C of 145 M−1s−1 and 5.8 M−1s−1, respectively. With GTN, the secondary nitrate was removed reductively 6 times faster than the primary nitrate, with liberation of nitrite. With propylene dinitrate, on the other hand, the primary nitrate residue was 3 times more reactive than the secondary residue. In the old yellow enzyme-catalyzed NADPH-dependent reduction of GTN and propylene dinitrate, ping-pong kinetics are displayed, as found for all other substrates of the enzyme. Rapid-reaction studies of mixing reduced enzyme with the nitrate esters show that a reduced enzyme–substrate complex is formed before oxidation of the reduced flavin. The rate constants for these reactions and the apparent Kd values of the enzyme–substrate complexes have been determined and reveal that the rate-limiting step in catalysis is reduction of the enzyme by NADPH. Analysis of the products reveal that with the enzyme-catalyzed reactions, reduction of the primary nitrate in both GTN and propylene dinitrate is favored by comparison with the free-flavin reactions. This preferential positional reactivity can be rationalized by modeling of the substrates into the known crystal structure of the enzyme. In contrast to the facile reaction of free reduced flavins with GTN, reduced 5-deazaflavins have been found to react some 4–5 orders of magnitude slower. This finding implies that the chemical mechanism of the reaction is one involving radical transfers.

7-Chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine S,S-dioxide (IDRA 21), a congener of aniracetam, potently abates pharmacologically induced cognitive impairments in patas monkeys.

We report here on the ability of IDRA 21 and aniracetam, two negative allosteric modulators of glutamate-induced DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization, to attenuate alprazolam-induced learning deficit in patas monkeys working in a complex behavioral task. In one component of a multiple schedule (repeated acquisition or "learning"), patas monkeys acquired a different four-response chain each session by responding sequentially on three keys in the presence of four discriminative stimuli (geometric forms or numerals). In the other component (performance) the four-response chain was the same each session. The response chain in each component was maintained by food presentation under a fixed-ratio schedule. When alprazolam (0.1 or 0.32 mg/kg p.o.) was administered alone, this full allosteric modulator of gamma-aminobutyric acid type A (GABAA) receptors produced large decreases in the response rate and accuracy in the learning component of the task. IDRA 21 (3 or 5.6 mg/kg p.o.) and aniracetam (30 mg/kg p.o.) administered 60 min before alprazolam, having no effect when given alone, antagonized the large disruptive effects of alprazolam on learning. From dose-response studies, it can be estimated that IDRA 21 is approximately 10-fold more potent than aniracetam in antagonizing alprazolam-induced learning deficit. We conclude that IDRA 21, a chemically unrelated pharmacological congener of aniracetam, improves learning deficit induced in patas monkeys by the increase of GABAergic tone elicited by alprazolam. Very likely IDRA 21 exerts its behavioral effects by antagonizing AMPA receptor desensitization.