No abstract

We evaluated several doses of cis-4-(phosphonomethyl)-2-piperidine-carboxylic acid (CGS-19755), a potent competitive N-methyl-D-aspartate (NMDA) receptor antagonist, systemically administered either before or after 20 to 30 minutes of global ischemia in rats. We measured outcome by mortality, histological damage by light microscopy, and learning ability on an eight-arm maze, and determined the drug's mechanism of action by an immunohistochemical assay of calcium-calmodulin binding. High-dose treatment begun prior to ischemia resulted in reduced cellular damage in severely ischemic hippocampal tissue, but also caused high mortality due to respiratory depression. Treatment begun 30 minutes after ischemia resulted in little histological protection but significantly improved learning ability when tested 1 month after ischemia, and did not increase mortality. Furthermore, CGS-19755, 10 mg/kg intraperitoneally, begun either before or after ischemia substantially reduced calcium influx into ischemic neurons as evidenced by reduced calcium-calmodulin binding. We conclude that CGS-19755 prevents calcium entry into ischemic neurons and may be effective therapy for very acute cerebral ischemia.

A cationic lipid-based gene delivery system composed of N- [(1-(2,3-dioleyloxy)propyl)]-N-N-N-trimethylammonium chloride and cholesterol, at a 4:1 molar ratio, was developed for systemic administration. Plasmid biodistribution and expression were characterized in syngeneic mouse tumor model squamous cell carcinoma VII cells. A reporter gene expression plasmid was used for biodistribution of plasmid and expression. The results showed that lungs and primary tumors were transfected. Fluorescence microscopy showed that fluorescent-labeled transfection complexes were passively targeted to the tumor vasculature and that the endothelial cells internalized the plasmid. Transgene expression was characterized based on duration of expression and dosing schedule. In vivo gene transfer with an interleukin-12 expression plasmid yielded protein levels in blood, lungs, and primary tumor after intravenous administration. Efficacy studies showed that 15 g of interleukin-12 plasmid was sufficient to produce a gene-specific inhibition of primary tumor growth. These results characterize the vascularity of the tumor model, characterize the in vivo gene transfer properties of the plasmid-based gene delivery system, and show that the transgene expression level was sufficient to elicit a biological response by inhibiting tumor growth.

Summ ary: Since ionic Ca 2 + binds with intracellular cal modulin (CaM) before activating proteases, kinases, and phospholipases, demonstration of persistent Ca 2 + -CaM binding in neurons destined to show ischemic cellular in jury would support the concept that elevated intracellular Ca2+ plays a causative role in ischemic neuronal damage. In order to characterize Ca 2 + -CaM binding, we used a sheep anti-CaM antibody (CaM-Ab) which recognizes CaM that is not bound to Ca 2 + or brain target proteins. Therefore, immunohistochemical staining of brain sec tions by labeled CaM-Ab represented only unbound CaM. Six normal rats were compared to 15 animals ren dered ischemic for 30 min by a modification of the four vessel occlusion model. Animals were killed immediately after ischemia, and after 2 and 24 h of reperfusion. Brain sections through hippocampus were incubated in CaM Ab, and a diaminobenzadiene labeled anti-sheep second ary antibody was added to stain the CaM-Abo Staining in the endal limb of dentate, dorsal CAl, lateral CA3, and parietal cortex was graded on a 4-point scale. All normal This study describes intraneuronal calcium calmodulin binding in a commonly used in vivo model of global cerebral ischemia. The project was undertaken for two reasons. It is now commonly accepted that calcium entry into neurons and re lease of stored intracellular calcium are pivotal events leading to irreversible cellular damage dur ing the reperfusion phase following an ischemic in sult. In the last several years many laboratories have demonstrated increased calcium in brain and in neurons after cerebral ischemia using a variety of

Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human alpha-transforming growth factor (alpha-TGF). The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting alpha-TGF. When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native alpha-TGF in conditioned cell culture media. When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species. Indirect immunofluorescence localization procedures revealed a high concentration of alpha-TGF in a perinuclear ring with a diffuse cytoplasmic distribution. These results suggest that a precursor form of alpha-TGF has a cellular role beyond that of an autocrine growth factor.