Improvement in Double Staining With Fluoro-Jade C and Fluorescent Immunostaining: FJC Staining Is Not Specific to Degenerating Mature Neurons

Fluoro-Jade C (FJC) staining has been used to detect degenerating neurons in tissue sections. It is a simple and easy staining procedure and does not depend on the manner of cell death. In some experiments, double staining with FJC and fluorescent immunostaining (FI) is required to identify cell types. However, pretreatment for FJC staining contains some processes that are harsh to fluorophores, and the FI signal is greatly reduced. To overcome this issue, we improved the double staining protocol to acquire clear double-stained images by introducing the labeled streptavidin–biotin system. In addition, several studies indicate that FJC can label non-degenerating glial cells, including resting/reactive astrocytes and activated microglia. Moreover, our previous study indicated that degenerating mesenchymal cells were also labeled by FJC, but it is still unclear whether FJC can label degenerating glial cells. Acute encephalopathy model mice contained damaged astrocytes with clasmatodendrosis, and 6-aminonicotinamide-injected mice contained necrotic astrocytes and oligodendrocytes. Using our improved double staining protocol with FJC and FI, we detected FJC-labeled degenerating astrocytes and oligodendrocytes with pyknotic nuclei. These results indicate that FJC is not specific to degenerating neurons in some experimental conditions:     

Plant growth-promoting oligosaccharides produced from tomato waste.

Tomato juice waste was hydrolyzed with acid. Tomato juice waste (500 g; wet weight) was heated with 0.5 N HCl (2.5 l) at 70 degrees C for 4 h. After neutralization, the growth-promoting extracts (300 g; dry weight) in the plants were produced from the tomato waste. The acid extract significantly promoted the growth of cockscomb (Celosia argentea L.) and tomato (Lycopersicon esculentum L.) seedlings. We have recognized potent plant growth-promoting substances in the acid extract from tomato waste. The most effective components in the active fraction were almost all oligogalacturonic acids (DP 6-12). This paper is the first report that plant growth-promoting oligosaccharides can be directly produced from tomato juice waste. It is possible that the substances from the tomato waste can become useful plant growth regulators in the agriculture field in the future.     

Excess Salt Increases Infarct Size Produced by Photothrombotic Distal Middle Cerebral Artery Occlusion in Spontaneously Hypertensive Rats

Cerebral circulation is known to be vulnerable to high salt loading. However, no study has investigated the effects of excess salt on focal ischemic brain injury. After 14 days of salt loading (0.9% saline) or water, spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were subjected to photothrombotic middle cerebral artery occlusion (MCAO), and infarct volume was determined at 48 h after MCAO: albumin and hemoglobin contents in discrete brain regions were also determined in SHR. Salt loading did not affect blood pressure levels in SHR and WKY. After MCAO, regional cerebral blood flow (CBF), determined with two ways of laser-Doppler flowmetry (one-point measurement or manual scanning), was more steeply decreased in the salt-loaded group than in the control group. In SHR/Izm, infarct volume in the salt-loaded group was 112±27 mm³, which was significantly larger than 77±12 mm³ in the control group (p = 0.002), while the extents of blood-brain barrier disruption (brain albumin and hemoglobin levels) were not affected by excess salt. In WKY, salt loading did not significantly increase infarct size. These results show the detrimental effects of salt loading on intra-ischemic CBF and subsequent brain infarction produced by phototrhombotic MCAO in hypertensive rats.     

Characterization of perdeuterated high-potential iron-sulfur protein with high-resolution X-ray crystallography

Perdeuteration in neutron crystallography is an effective method for determining the positions of hydrogen atoms in proteins. However, there is shortage of evidence that the high-resolution details of perdeuterated proteins are consistent with those of the nondeuterated proteins. In this study, we determined the X-ray structure of perdeuterated high-potential iron-sulfur protein (HiPIP) at a high resolution of 0.85 å resolution. The comparison of the nondeuterated and perdeuterated structures of HiPIP revealed slight differences between the two structures. The spectroscopic and spectroelectrochemical studies also showed that perdeuterated HiPIP has approximately the same characteristics as nondeuterated HiPIP. These results further emphasize the suitability of using perdeuterated proteins in the high-resolution neutron crystallography.