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Lipocortin 1 (LC1, annexin 1) has received considerable attention as a substrate for protein kinases, as a Ca++- and phosphatidylserine-binding protein, and as a mediator of glucocorticoid anti-inflammatory effects. However, there has been confusion over localization of LC1 immunoreactivity (LC1-ir), which reportedly localizes to neurons and/or to astrocytes or microglia in rat brain. To test whether these contradictory data arise from unusual properties of the antigen, we developed a novel brain slice model to determine fixation and staining variables. The specificity of anti-LC1 sera was ensured by pre-absorption and affinity purification with immobilized recombinant LC1. Specific LC1-ir was detected in ramified microglia of brains perfused with acidified aldehydes and embedded in paraffin. However, commonly used immunohistochemical procedures have unexpected profound effects. LC1-ir was eliminated by fixation with neutral/alkaline aldehydes, by freezing before strong acid-aldehyde fixation, or by staining without partial de/rehydration before the primary serum. The sensitivity of LC1 epitopes to proton and water activities may reflect molecular properties important to LC1's roles in vivo. True LC1-ir was not detected in normal neurons or astrocytes.
The recently developed controlled cortical impact model of brain injury in rats may be an excellent tool by which to attempt to understand the neurochemical mechanisms mediating the pathophysiology of traumatic brain injury. In this study, rats were subjected to lateral controlled cortical impact brain injury of low grade severity; their brains were frozen in situ at various times after injury to measure regional levels of lactate, high energy phosphates, and norepinephrine. Tissue lactate concentration in the injury site left cortex was increased in injured animals by sixfold at 30 min and twofold at 2.5 h and 24 h after injury (p < 0.05). At all postinjury times, lactate concentration was also increased in injured animals by about twofold in the cortex and hippocampus adjacent to the injury site (p < 0.05). No significant changes occurred in the levels of ATP and phosphocreatine in most of the brain regions of injured animals. However, in the primary site of injury (left cortex), phosphocreatine concentration was decreased by 40% in injured animals at 30 min after injury (p < 0.05). The norepinephrine concentration was decreased in the injury site left cortex of injured animals by 38% at 30 min, 29% at 2.5 h, and 30% at 24 h after injury (p < 0.05). The level of norepinephrine was also reduced by approximately 20% in the cortex adjacent to the injury site in injured animals. The present results suggest that controlled cortical impact brain injury produces disorder in the neuronal oxidative and norepinephrine metabolism.
We have used tobacco mosaic virus (TMV) as a test specimen, in order to develop techniques for the analysis of high-resolution structural detail in electron micrographs of biological assemblies with helical symmetry. It has previously been shown that internal details of protein structure can be visualized by processing electron micrographs of unstained specimens of extended two-dimensional crystalline arrays. However, the techniques should in principle be applicable to other periodic specimens, such as assemblies with helical symmetry. We show here that data to spacings better than 10 A can be retrieved from electron images of frozen hydrated TMV. The three-dimensional computed map agrees well with that derived from X-ray diffraction and shows the two pairs of alpha-helices forming the core of the coat subunit, the C alpha-helix and the viral RNA. The results demonstrate that it is possible to determine detailed internal structure in helical particles.