Immunohistochemical localization of mitochondrial fatty acid ß-oxidation enzymes in Müller cells of the retina.

The presence of a mitochondrial fatty acid ß-oxidation system in the retina was shown by immunohistochemistry. Fatty acids are considered to serve as a major energy source metabolized by fatty acid ß-oxidation together with glucose metabolized by glycolysis in the organs of the entire body, but almost nothing is known about this metabolic system in the retina. Adult rat retinae were subjected to immunofluorescence and immuno-electron microscopy for the localization of fatty acid ß-oxidation enzymes, together with western blot analysis for quantitation of the amount of enzyme proteins and DNA microarray analysis for gene expression. All the enzymes examined were shown to be present in the retina, but in small amounts, with the amount of protein and gene expression in the retina being about 1/10 of those in the liver. Immunohistochemistry at light and electron microscopic levels revealed the enzymes to be more preferentially localized to the mitochondria of Müller cells than the retinal neurons. The Müller cells were isolated from the retina and confirmed for the presence of mitochondrial fatty acid ß-oxidation enzymes. A mitochondrial fatty acid ß-oxidation system was thus shown to be present in the retina heterogeneously.

Immunohistochemical localization of mitochondrial fatty acid ß-oxidation enzymes in rat testis.

The testis consists of two types of tissues, the interstitial tissue and the seminiferous tubule, which have different functions and are assumed to have different nutritional metabolism. The localization of enzymes of the mitochondrial fatty acid ß-oxidation system in the testis was investigated to obtain a better understanding of nutrient metabolism in the testis. Adult rat testis tissues were subjected to immunoblot analysis for quantitation of the amounts of enzyme proteins, to DNA microarray analysis for gene expression, and to immunofluorescence and immunoelectron microscopy for localization. Quantitative analysis by immunoblot and DNA microarray revealed that enzymes occur abundantly in Leydig cells in the interstitial tissue but much less so in the seminiferous tubules. Immunohistochemistry revealed that Leydig cells in the interstitial tissue and Sertoli cells in the seminiferous tubules contain a full set of mitochondrial fatty acid ß-oxidation enzymes in relatively plentiful amounts among the cells in the testis, but that this is not so in spermatogenic cells. This characteristic localization of the mitochondrial fatty acid ß-oxidation system in the testis needs further elucidation in terms of a possible role for it in the nutritional metabolism of spermatogenesis.

High-contrast imaging of plastic-embedded tissues by phase contrast electron microscopy.

Phase contrast electron microscopy utilizing phase plates has been considered suitable for high-contrast observation of weak phase objects. This novel technique was newly applied to histochemically stained strong phase objects of osmificated biological specimens. Sections of various thicknesses, specifically stained for the Golgi apparatus by the ZIO technique using the heavy metals Zn and Os, were observed with a phase contrast electron microscope in Zernike and Hilbert imaging modes. Quantitative analysis of image contrast in real space and the power spectrum in Fourier space showed a high-contrast gain even for strong phase objects. This result clearly indicates that phase contrast electron microscopy can be effectively used not only for weak phase objects but also for strong phase objects in biology.

Activation of the JAK-STAT signaling pathway in the rat basilar artery after subarachnoid hemorrhage.

The Janus kinase-signal transducer and activator of transcription (JAK-STAT) is one of the most important signaling pathways transducing signals from the cell surface in response to cytokines. Subarachnoid hemorrhage (SAH) produces cytokines in the CSF. We investigated whether this signaling pathway is activated in the rat basilar artery after SAH by cytokines. In a rat single-hemorrhage model of SAH, basilar arteries and CSF were obtained until 7 days after SAH. The concentration of interleukin-6 (IL-6) in CSF was measured by ELISA. Western blot analysis with JAK1, phosphospecific-JAK1, STAT3, phosphospecific STAT3 at Tyr705 and Ser727, cyclooxygenase-2 (COX-2), and actin antibodies was performed in basilar artery. The expressions of STAT3, phosphospecific STAT3 at Tyr705 and Ser727, and COX-2 in basilar artery were examined by immunohistochemical studies. The concentration of IL-6 immediately increased after SAH and Western blot analysis revealed that JAK1 was phosphorylated within 2 h, accompanied by phosphorylation of STAT3 at Tyr705, extending to Ser727 at days 1-2. Immunohistochemistry revealed phosphorylation of STAT3 to occur in endothelial and smooth muscle cells of the basilar artery. In addition, intracisternal injection of IL-6 by itself significantly increased phosphorylation of STAT3 at Tyr705 and Ser727. Expression of COX-2 was also upregulated in endothelial cells of the basilar artery. These results indicate that SAH produces the proinflammatory cytokine IL-6 in the CSF, which activates the JAK-STAT signaling pathway in the basilar artery and induces transcription of immediate early genes.

Activation of JAK/STAT signalling in neurons following spinal cord injury in mice.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway is one of the most important in transducing signals from the cell surface to the nucleus in response to cytokines. In the present study, we investigated chronological alteration and cellular location of JAK1, STAT3, phosphorylated (p)-Tyr1022/1023-JAK1, p-Tyr705-STAT3, and interleukin-6 (IL-6) following spinal cord injury (SCI) in mice. Western blot analysis showed JAK1 to be significantly phosphorylated at Tyr1022/1023 from 6 h after SCI, peaking at 12 h and gradually decreasing thereafter, accompanied by phosphorylation of STAT3 at Tyr705 with a similar time course. ELISA analysis showed the concentration of IL-6 in injured spinal cord to also significantly increase from 3 h after SCI, peaking at 12 h, then gradually decreasing. Immunohistochemistry revealed p-Tyr1022/1023-JAK1, p-Tyr705-STAT3, and IL-6 to be mainly expressed in neurons of the anterior horns at 12 h after SCI. Pretreatment with a JAK inhibitor, AG-490, suppressed phosphorylation of JAK1 and STAT3 at 12 h after SCI, reducing recovery of motor functions. These findings suggest that SCI at the acute stage produces IL-6 mainly in neurons of the injured spinal cord, which activates the JAK/STAT pathway, and that this pathway may be involved with neuronal response to SCI.

Modification of endothelial NO synthase through protein phosphorylation after forebrain cerebral ischemia/reperfusion.

BACKGROUND AND PURPOSE: Production of NO by endothelial NO synthase (eNOS) is thought to play a neuroprotective role after cerebral ischemia. The vascular endothelial growth factor (VEGF) contributes to activation of eNOS by Ca2+/calmodulin and also stimulates the protein kinase Akt, which directly phosphorylates eNOS on Ser1177 and increases enzyme activity. Although the expression of VEGF has been studied in ischemic stroke models, the activation of eNOS after cerebral ischemia has not been investigated. The purpose of the present study was to clarify molecular mechanisms underlying the regulation of eNOS activity through protein phosphorylation in postischemic processes. METHODS: Sprague-Dawley rats were subjected to forebrain cerebral ischemia for 15 minutes with hypotension and reperfusion for up to 24 hours. Western blot analysis and ELISAs were used to study the temporal profiles of Akt, phospho-Akt at Ser437, eNOS, phospho-eNOS at Ser1177, and VEGF expression, respectively. Immunohistochemical studies were performed to examine the spatial expression patterns of phospho-Akt at Ser437 and phospho-eNOS at Ser1177. RESULTS: Increase in phospho-Akt at Ser437 was observed transiently 0.5 to 2 hours after reperfusion, whereas elevation of phospho-eNOS at Ser1177 and VEGF expression was observed from 6 hours after reperfusion. Endothelial cells in the microvessels were the major source of eNOS phosphorylated at Ser1177 at the 12-hour time point. CONCLUSIONS: Increase in Ser1177 phospho-eNOS occurs in endothelial cells of microvessels after ischemic episodes with temporal expression of VEGF, pointing to a contribution to the autoregulation of postischemic brain damage.

Localization of translational components at the ultramicroscopic level at postsynaptic sites of the rat brain.

We investigated the localization of components of translational machinery and their regulators in the postsynaptic region. We examined several components, especially those involved in translational regulation: components of (1) MAPK-Mnk-eIF4E, (2) PI3-kinase-PDK-Akt/PKB-FRAP/mTOR-PHAS/4EBP, (3) p70S6K-S6 ribosomal protein and (4) eEF2 kinase/CaMKIII-eEF2 pathways. Western blotting detected all the components examined in the synaptic fractions, and their differential localization to the synaptic subcompartments: initiation or elongation factors, except for eIF5, were detected predominantly in the dendritic lipid raft fraction, which contained ER marker proteins. In contrast, most of their regulatory kinases were distributed to both the postsynaptic density (PSD) and the dendritic lipid raft fractions, or enriched in the former fraction. Localization of eIF4E at synaptic sites was further examined immunohistochemically at the electron microscopic level. The eIF-4E-immunoreactivity was localized to the postsynaptic sites, especially to the microvesicle-like structures underneath the postsynaptic membrane in the spine, some of which were localized in close proximity to PSD. These results suggest that the postsynaptic local translational system, in at least four major regulatory pathways, is similar to those in the perinuclear one, and that it takes place, at least partly, immediately beneath the postsynaptic membrane. The results also suggest the presence of ER-associated type of translational machinery at the postsynaptic sites.

Phosphorylation of neuronal nitric oxide synthase at Ser847 by CaM-KII in the hippocampus of rat brain after transient forebrain ischemia.

The authors previously demonstrated that Ca2+/calmodulin (CaM)-dependent protein kinase IIalpha (CaM-KIIalpha) can phosphorylate neuronal nitric oxide synthase (nNOS) at Ser847 and attenuate NOS activity in neuronal cells. In the present study, they established that forebrain ischemia causes an increase in the phosphorylation of nNOS at Ser847 in the hippocampus. This nNOS phosphorylation appeared to be catalyzed by CaM-KII: (1) it correlated with the autophosphorylation of CaM-KIIalpha; (2) it was blocked by the CaM-KII inhibitor, KN-93; and (3) nNOS and CaM-KIIalpha were found to coexist in the hippocampus. Examination of the spatial relation between nNOS and CaM-KIIalpha in the brain revealed coexistence in the hippocampus but not in the cortex during reperfusion, with a concomitant increase in autophosphorylation of CaM-KIIalpha. The phosphorylation of nNOS at Ser847 probably takes place in nonpyramidal hippocampal neurons, which increased after 30 minutes of reperfusion in the hippocampus, whereas no significant increase was detected in the cortex. An intraventricular injection of KN-93 significantly decreased the phosphorylation of nNOS in the hippocampus. These results point to CaM-KII as a protein kinase, which by its colocalization may attenuate the activity of nNOS through its Ser847 phosphorylation, and may thus contribute to promotion of tolerance to postischemic damage in hippocampal neurons.