Translational attenuation of the Bacillus subtilis spo0B cistron by an RNA structure encompassing the initiation region.

The spo0B gene, which exists as an operon with the obg gene, is required to initiate sporulation (stage 0) of Bacillus subtilis . This gene encodes a phosphotransferase in the multicomponent phosphorelay system. We here report the novel finding that a spo0B 5'-terminal SLR (stem-loop structure sequestering ribosome binding sequence; ACUCCUAA-X16-UUG GGAG U, Delta G = -8.71 kcal/mol) attenuated spo0B translation. The spo0B gene was efficiently transcribed but Spo0B protein was not overproduced in Escherichia coli when spo0B was induced using expression vectors carrying the SLR- spo0B region under control of the tac promoter. Deletion of the SLR from the vectors resulted in overexpression of spo0B . Therefore, to characterize expression of spo0B with a SLR in B.subtilis we constructed transcriptional and translational lacZ fusions combined with the spo0B 5'-terminal region with a deleted or mutagenized SLR. These constructs were subsequently introduced into B.subtilis as multiple and single copies, then beta-galactosidase activities were measured. The possible SLR also functioned as a negative cis element in B.subtilis. Furthermore, B.subtilis strain 1S16 (spo0B136) lysogenized straight phiCD0B-S and -W, harboring spo0B with mutagenized SLRs that were more (Delta G = -14.0 kcal/mol) and less-stable (Delta G = -1.31 kcal/mol) compared with the wild-type, exhibited null and wild-type sporulation respectively. These results indicate that the spo0B 5'-SLR affects spo0B gene expression for sporulation but that low expression of spo0B through the wild-type SLR was sufficient to initiate sporulation in B.subtilis.

Delayed neuronal death prevented by inhibition of increased hydroxyl radical formation in a transient cerebral ischemia.

The salicylate-trapping method was used to detect hydroxyl radicals by measurement of stable adduct dihydroxybenzoic acid (DHBA). Ten minutes of forebrain ischemia followed by reperfusion induced the increase in DHBA in rat hippocampal perfusates. Postischemic treatment with a free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), significantly reduced the increase in DHBA and suppressed delayed neuronal death in the hippocampal CA1 region.

Effects of a novel free radical scavenger, MCl-186, on ischemic brain damage in the rat distal middle cerebral artery occlusion model.

We investigated the effects of a free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCl-186), on infarct areas, neurological deficits and regional cerebral blood flow (rCBF), with use of a rat thrombotic distal middle cerebral artery (dMCA) occlusion model to elucidate its possible therapeutic effects on focal cerebral ischemia. In addition, we have attempted to measure 2-oxo-3-(phenylhydrazono)-butanoic acid (OPB), which is the major oxidation product of MCl-186, in the penumbral cortex of a thrombotic dMCA occlusion model. Postischemic treatment with MCl-186 (3 mg/kg) significantly (P < .05) decreased the size of the cerebral infarcts 1 day after dMCA occlusion. MCl-186 (3 mg/kg) significantly (P < .05) improved the neurological deficits 1 day after dMCA occlusion. On the contrary, MCl-186 had no effect on rCBF 1 day after dMCA occlusion. MCl-186 mainly reacted into OPB by peroxidation in rat brain homogenates. Furthermore, the increase in OPB content in the ischemic penumbral cortex tissue was confirmed after 90 min of MCl-186 perfusion. These results suggest that MCI-186 has a protective effect on brain ischemia by reacting with oxygen radicals and that oxygen radicals are closely related to postischemic brain injury.

Calcium-activated neutral proteinase (calpain) system in aging and Alzheimer's disease.

Calpains (CANPs) are a family of calcium-dependent cysteine proteases under complex cellular regulation. By making selective limited proteolytic cleavages, they activate or alter the regulation of certain enzymes, including key protein kinases and phosphatases, and induce specific cytoskeletal rearrangements, accounting for their suspected involvement in intracellular signaling, vesicular trafficking, and structural stabilization. Calpain activity has been implicated in various aging phenomena, including cataract formation and erythrocyte senescence. Abnormal activation of the large stores of latent calpain in neurons induces cell injury and is believed to underlie neurodegeneration in excitotoxicity, Wallerian degeneration, and certain other neuropathologic states involving abnormal calcium influx. In Alzheimer's disease, we found the ratio of activated calpain I to its latent precursor isoform in neocortex to be threefold higher than that in normal individuals and those with Huntington's or Parkinson's disease. Immunoreactivity toward calpastatin, the endogenous inhibitor of calpain, was also markedly reduced in layers II-V of the neocortex in Alzheimer's disease. The excessive calpain system activation suggested by these findings represents a potential molecular basis for synaptic loss and neuronal cell death in the brain in Alzheimer's disease given the known destructive actions of calpain I and its preferential neuronal and synaptic localization. In surviving cells, persistent calpain activation may also contribute to neurofibrillary pathology and abnormal amyloid precursor protein trafficking/processing through its known actions on protein kinases and the membrane skeleton. The degree of abnormal calpain activation in the brain in Alzheimer's disease strongly correlated with the extent of decline in levels of secreted amyloid precursor protein in brain. Cytoskeletal proteins that are normally good calpain substrates become relatively calpain resistant when they are hyperphosphorylated, which may contribute to their accumulation in neurofibrillary tangles. As a major effector of calcium signals, calpain activity may mirror disturbances in calcium homeostasis and mediate important pathologic consequences of such disturbances.

Immunoassay and activity of calcium-activated neutral proteinase (mCANP): distribution in soluble and membrane-associated fractions in human and mouse brain.

The millimolar form of calcium-activated neutral proteinase (mCANP) is generally regarded as a cytosolic enzyme in nonneuronal systems, although its subcellular localization in brain is less well established. To resolve conflicting reports on the localization of mCANP based on activity measurements, we developed an immunoassay for CANP and compared the content and activity of the molecule in soluble and membrane fractions of mouse and human brain. Western blot immunoassays, using two different antibodies specific for mCANP, demonstrated that mCANP content is 4.5 ng/g in human or mouse brain, about 0.0005% of the total protein. More than 95% of the total immunoreactive mCANP remained in the soluble fraction after 15,000 g centrifugation of the whole homogenate. mCANP activity was determined with [14C]azocasein as substrate after removing endogenous CANP inhibitor(s) by ion-exchange chromatography on DEAE-cellulose. Caseinolytic activity was detected only in fractions derived from the supernatant extract. The distribution of mCANP content and enzyme activity were unchanged when tissues were extracted with different concentrations of Triton X-100. These findings establish the usefulness and validity of the CANP immunoassay and demonstrate that mCANP in mouse and human brain is localized predominantly within the cytosol.

Changes of electroretinogram and neurochemical aspects of GABAergic neurons of retina after intraocular injection of kainic acid in rats.

The effect of kainic acid (KA) on both electroretinogram (ERG) readings and neurochemical properties of the retina was investigated in rats with emphasis placed upon examination of the events that occur immediately following KA treatment. KA was injected into the eyes of rats with doses of 50 and 200 nmol. One hour after injection, histological alterations became evident. Swelling was observed in the inner and outer plexiform layers. Certain ganglion cells and cells of the inner nuclear layers exhibited pyknotic nuclei. Most of the ganglion cells appeared to have degenerated 48 h following injection, and the form of the outer plexiform layer was incomplete. The amplitude of the b-waves of the ERG decreased 2 h following injection and never recovered. The amplitude of the a-waves was unaffected by KA. The gamma-aminobutyric acid content in the eyecups began to decrease within 1 h and fell to approximately 20% of its original level 24 h following injection. The taurine content in the eyecups was unaffected by KA. The activity of glutamic acid decarboxylase remained unaffected for 2 h after injection, but was reduced to approximately 40% of its original activity by 24 h after injection. A possible explanation for the mechanism by which KA effects degenerative changes in the rat retina is that KA induces release of neurotransmitters through stimulation of neurons, and degeneration in the soma follows.