Expression of the inducible nitric oxide synthase in distinct cellular types after traumatic brain injury: an in situ hybridization and immunocytochemical study.

The Marmarou's acceleration traumatic brain injury (TBI) model, in situ hybridization and immunocytochemistry were utilized to study the temporal expression of the inducible form of nitric oxide synthase (iNOS) mRNA and protein in different cellular compartments of the rat brain. Four hours following TBI, expression of iNOS was observed in the endothelial cells of cerebral blood vessels, macrophages and many cortical and hippocampal neurons. In the cortex labeled neuronal and nonneuronal cells were primarily found in the superficial layers. In the hippocampus the strongest neuronal labeling was observed in the CAI and CA3 (lateral part) regions. By 24 h post TBI endothelial cells no longer expressed iNOS mRNA, and the macrophage and neuronal iNOS expression was reduced by 30-50%. The reduction was assessed by automated quantitation of the silver grains that occupy individual cellular profiles using an image analysis system. Immunocytochemistry revealed de novo iNOS synthesis in non-neuronal cells at the different time points, thus paralleling the changes in iNOS mRNA expression. In contrast, iNOS immunoreactivity in neurons was not observed before 24 h post TBI, suggesting failure of iNOS protein translation at 4 h after trauma. The results demonstrate complex spatial and temporal patterns of iNOS expression in discrete cellular populations, indicating different times of nitric oxide synthesis (and release) following TBI. Uncoupling of iNOS mRNA and protein synthesis in neurons suggests differential synthesis of nitric oxide in these cells as compared to non-neuronal cellular populations after trauma.

Ultrastructural localization of phosphorylated eIF2alpha [eIF2alpha(P)] in rat dorsal hippocampus during reperfusion.

During post-ischemic brain reperfusion there is a substantial reduction of protein synthesis in selectively vulnerable neurons. Normal protein synthesis requires a functional translation initiation complex, a key element of which is eukaryotic initiation factor 2 (eIF2), which in a complex with GTP introduces the met-tRNA(i). Phosphorylation of Ser(51) on the alpha subunit of eIF2 [eIF2alpha(P)] generates a competitive inhibitor of eIF2B, thereby preventing the replenishment of GTP onto eIF2, thus blocking translation initiation. It has been shown that the conditional expression of an eIF2alpha mutant (Asp substituted for Ser(51)) imitating the negative charge of Ser(51) (P) induces apoptosis. During the first 10 min of post-ischemic reperfusion, there is an approximately 20-fold increase in eIF2alpha(P) seen in the cytoplasm of CA1 hippocampal neurons, and, by 1 h, there is also accumulation of eIF2alpha(P) in the nucleus. We utilized post-embedding electron microscopical immunogold methods to examine the localization of eIF2alpha(P) during reperfusion. Immunogold particles (10 nm) were concentrated chiefly along the rough endoplasmic reticulum and in association with the membranes of the nuclear envelope in CA1 neurons. Aggregations of gold particles in the nucleus were concentrated: (1) within and around the nucleolus, (2) associated to strands of heterochromatin, and (3) along putative nuclear filaments. The presence of eIF2alpha(P) in the nucleolus probably reflects its association with nascent ribosomal subunits. The beta-subunit of eIF2 has a zinc finger and polylysine blocks analogous to those on other proteins that affect transcription. The association of eIF2alpha(P) with chromatin may have important implications for transcription.

Enigmatic resistance of sheep (Ovis aries) to infection by virulent Francisella tularensis.

Tularemia in range sheep is an occasional cause of severe economic loss from mortality and unthriftiness as well as a hazard to persons in contact with the animals. Epizootics are unpredictable and explosive, therefore, prophylaxis is more practical than therapy. Live vaccine of proven value in man and in beavers was inoculated into mature ewes and elicited antibodies without harm to the sheep. However, challenge of immunity was not possible because virulent Francisella tularensis in large doses did not cause significant mortality in healthy, well managed, unimmunized sheep. Evidence suggests that a complex of stresses such as inclement weather, lambing and concomitant ectoparasitism render sheep more susceptible to tularemia.

Yersinia philomiragia sp. n., a new member of the Pasteurella group of bacteria, naturally pathogenic for the muskrat (Ondatra zibethica).

A bacterium experimentally pathogenic for muskrats (Ondatra zibethica), white mice, mountain voles (Microtus montanus), and deer mice (Peromyscus maniculatus) was isolated from the tissues of a sick muskrat captured on the Bear River Migratory Bird Refuge (Brigham City, Utah) and from four surface water samples collected within 15 miles of that point. In culture, the cells are chiefly coccoid, but in the tissues of muskrats and voles they resemble the bizarre forms of Yersinia pestis, except for their smaller size. The characteristics of the organism are described and the name Yersinia philomiragia sp. n. is proposed.