scholarly journals The Effect of Brain Homogenates on Directly Measured Water Fluxes Through The Pronotum of Periplaneta Americana

1992 ◽  
Vol 171 (1) ◽  
pp. 395-408
Author(s):  
J. MACHIN ◽  
P. KESTLER ◽  
G. J. LAMPERT
Keyword(s):  
Periplaneta Americana ◽  
Tritiated Water ◽  
Brain Homogenate ◽  
Permeability Change ◽  
Water Fluxes ◽  
Brain Extracts ◽  
Order Of Magnitude ◽  
The Brain ◽  
Measured Water

Measurements of tritiated water (THO) efflux using a cup, ventilated with dry air, attached to the pronotum of Periplaneta, have been used to obtain unambiguous measurements of the permeability of this structure. Permeability values were 53% of those determined gravimetrically. Our results support the proposal that cockroach cuticle permeability is hormonally controlled, to the extent that compounds extracted from the brain have been shown to be capable of inducing permeability change over relatively short periods. Fresh brain homogenate injections from hydrated donors produced a 28% increase in pronotal permeability in 5 h, rising to 46% the day after. Injections of saline or of fresh brain extracts from dehydrated and normally hydrated cockroaches had no effect. However, previously frozen brain homogenates, from donors at all hydration levels, significantly increased pronotal permeability the day following injection. The capacity of the pronotum to undergo increases in permeability over relatively short periods was also evident in other experiments. THO permeabilities of excised pronotal discs (5.16×10−10±0.31×10−10ms−1) were an order of magnitude higher than those of intact pronota (0.49×10−10±0.06×10−10 ms−1). In vivo permeability of pronota of accidentally injured cockroaches was significantly higher than that of uninjured animals, even though the pronota themselves were not damaged. We argue that the observed changes in cuticle permeability are too small primarily to serve osmoregulatory functions. We suggest, instead, that the changes might be associated with the control of secondary processes in which the cuticle is involved and which unavoidably promote water loss.

scholarly journals Neuronal spreading and plaque induction of intracellular Aβ and its disruption of Aβ homeostasis

2021 ◽  
Author(s):  
Tomas T. Roos ◽  
Megg G. Garcia ◽  
Isak Martinsson ◽  
Rana Mabrouk ◽  
Bodil Israelsson ◽  
...  
Keyword(s):  
Brain Homogenate ◽  
Fold Increase ◽  
Amyloid Beta Peptide ◽  
Intracellular Accumulation ◽  
Brain Areas ◽  
Cellular Models ◽  
Beta Peptide ◽  
The Brain

AbstractThe amyloid-beta peptide (Aβ) is thought to have prion-like properties promoting its spread throughout the brain in Alzheimer’s disease (AD). However, the cellular mechanism(s) of this spread remains unclear. Here, we show an important role of intracellular Aβ in its prion-like spread. We demonstrate that an intracellular source of Aβ can induce amyloid plaques in vivo via hippocampal injection. We show that hippocampal injection of mouse AD brain homogenate not only induces plaques, but also damages interneurons and affects intracellular Aβ levels in synaptically connected brain areas, paralleling cellular changes seen in AD. Furthermore, in a primary neuron AD model, exposure of picomolar amounts of brain-derived Aβ leads to an apparent redistribution of Aβ from soma to processes and dystrophic neurites. We also observe that such neuritic dystrophies associate with plaque formation in AD-transgenic mice. Finally, using cellular models, we propose a mechanism for how intracellular accumulation of Aβ disturbs homeostatic control of Aβ levels and can contribute to the up to 10,000-fold increase of Aβ in the AD brain. Our data indicate an essential role for intracellular prion-like Aβ and its synaptic spread in the pathogenesis of AD.

scholarly journals A study on Dowager Cixi’s Yanling-Yishou-Dan of Qing Dynasty in a Japanese laboratory of biochemistry and molecular biology in 1990s: An attempt for TCM modernization

2021 ◽  
pp. 1-21
Author(s):  
Jiankang Liu
Keyword(s):  
Qing Dynasty ◽  
Brain Homogenate ◽  
Modern Technology ◽  
Toxicity Tests ◽  
Brain Functions ◽  
Great Progress ◽  
Theoretical Evidence ◽  
The Brain

Traditional Chinese Medicine (TCM) modernization has been proposed for many years, but the progress is still slow due to both ideological and technical obstacles. When I went to Japan in 1989, I found Japan has made a great progress on TCM by using modern technology. Therefore, I have studied a fine extract prepared from medicinal herbs (renamed Yi-Zhi-Yi-Shou, YZYS), a prescription of Dowager Cixi’s Yanling-Yishou-Dan of Qing Dynasty, with the current drug investigation strategies. I examined its antioxidant activity both in vitro and in vivo. The in-vitro studies found that YZYS possesses strong antioxidant capacity, such as scavenging various kinds of free radicals, and inhibits free radical-induced peroxidation of brain homogenate, microsomes, mitochondria, amino acids, deoxyribose and DNA. The in-vivo study with immobilization-induced emotional stress in rats, showed that YZYS effectively inhibits stress-induced stomach ulcers and oxidative damage in plasma and the brain. In addition, YZYS is shown to be non-toxic in both acute and chronic toxicity tests. These studies demonstrate that YZYS is a potent natural antioxidant and offer theoretical evidence for the beneficial effect of YZYS on health and brain functions, and that TCM prescriptions can be studied scientifically as modern medical drugs.

scholarly journals The Retention Mechanism of Technetium-99m-HM-PAO: Intracellular Reaction with Glutathione

1988 ◽  
Vol 8 (1_suppl) ◽  
pp. S4-S12 ◽  
Author(s):  
Rudi D. Neirinckx ◽  
James F. Burke ◽  
Roger C. Harrison ◽  
Alan M. Forster ◽  
Allan R. Andersen ◽  
...  
Keyword(s):  
Steady State ◽  
Tissue Distribution ◽  
Rat Brain ◽  
Rate Constant ◽  
Conversion Rate ◽  
Brain Homogenate ◽  
Retention Mechanism ◽  
Technetium 99M ◽  
The Brain

Preparations of d,l- and meso-hexamethylpropyleneamine oxime (HM-PAO) labeled with technetium-99m were added to rat brain homogenates diluted with phosphate buffer (l: 10). The conversion of d,l-HM-PAO to hydrophilic forms took place with an initial rate constant of 0.12 min−1. Incubation of the brain homogenate with 2% diethyl maleate for 5 h decreased the homogenate's measured glutathione (GSH) concentration from 160 to 16 μ M and decreased the conversion rate to 0.012 min−1. Buffered aqueous solutions of glutathione rapidly converted the HM-PAO tracers to hydrophilic forms having the same chromatographic characteristics as found in the brain homogenates. The rate constant for the conversion reaction of d,l-HM-PAO in GSH aqueous solution was 208 and 317 L/mol/min in two different assay systems and for meso-HM-PAO the values were 14.7 and 23.2 L/mol/min, respectively. Rat brain has a GSH concentration of about 2.3 m M and the conversion of the d,l-HM-PAO due to GSH alone should proceed with a rate constant of 0.48 to 0.73 min−1 and be correspondingly 14-fold slower for meso-HM-PAO. In human brain, the in vivo data of Lassen et al. show a conversion rate constant of 0.80 min−1. This correspondence of values supports the notion that GSH may be important for the in vivo conversion of 99mTc-labeled HM-PAO to hydrophilic forms and may be the mechanism of trapping in brain and other cells. A kinetic model for the trapping of d,l- and meso-HM-PAO in tissue is developed that is based on data of GSH concentration in various organs. This model predicts that the d,l form rapidly reaches a steady state in tissue and the tissue distribution reflects a pattern dominated by blood flow. For the meso form, the model predicts that steady state is reached more slowly and the tissue distribution reflects a pattern dominated by glutathione concentration.

scholarly journals A 11C-Labeled 1,4-Dihydroquinoline Derivative as a Potential PET Tracer for Imaging of Redox Status in Mouse Brain

2015 ◽  
Vol 35 (12) ◽  
pp. 1930-1936 ◽  
Author(s):  
Toshimitsu Okamura ◽  
Maki Okada ◽  
Tatsuya Kikuchi ◽  
Hidekatsu Wakizaka ◽  
Ming-Rong Zhang
Keyword(s):  
Mouse Brain ◽  
Initial Velocity ◽  
Brain Homogenate ◽  
Redox Balance ◽  
Redox Status ◽  
Cationic Form ◽  
Pet Tracer ◽  
Positron Emission ◽  
The Brain

A disturbance in redox balance has been implicated in the pathogenesis of a number of diseases. This study sought to examine the feasibility of imaging brain redox status using a 11C-labeled dihydroquinoline derivative ([11C]DHQ1) for positron emission tomography (PET). The lipophilic PET tracer [11C]DHQ1 was rapidly oxidized to its hydrophilic form in mouse brain homogenate. The redox modulators diphenyleneiodonium and apocynin significantly reduced the initial velocity of [11C]DHQ1 oxidation, and apocynin also caused concentration-dependent inhibition of the initial velocity. Moreover, [11C]DHQ1 readily entered the brain by diffusion after administration and underwent oxidation into the hydrophilic cationic form, which then slowly decreased. By contrast, apocynin treatment inhibited the in vivo oxidation of [11C]DHQ1 to the hydrophilic cationic form, leading to a rapid decrease of radioactivity in the brain. Thus, the difference in the [11C]DHQ1 kinetics reflects the alteration in redox status caused by apocynin. In conclusion, [11C]DHQ1 is a potential PET tracer for imaging of redox status in the living brain.

scholarly journals Synapsin I: an actin-bundling protein under phosphorylation control.

1987 ◽  
Vol 105 (3) ◽  
pp. 1355-1363 ◽  
Author(s):  
T C Petrucci ◽  
J S Morrow
Keyword(s):  
Actin Filaments ◽  
Binding Activity ◽  
Brain Extract ◽  
Synapsin I ◽  
Peptide Fragments ◽  
Molecular Weights ◽  
Brain Extracts ◽  
The Brain ◽  
Precise Role

Synapsin I is a neuronal phosphoprotein comprised of two closely related polypeptides with apparent molecular weights of 78,000 and 76,000. It is found in association with the small vesicles clustered at the presynaptic junction. Its precise role is unknown, although it probably participates in vesicle clustering and/or release. Synapsin I is known to associate with vesicle membranes, microtubules, and neurofilaments. We have examined the interaction of purified phosphorylated and unphosphorylated bovine and human synapsin I with tubulin and actin filaments, using cosedimentation, viscometric, electrophoretic, and morphologic assays. As purified from brain homogenates, synapsin I decreases the steady-state viscosity of solutions containing F-actin, enhances the sedimentation of actin, and bundles actin filaments. Phosphorylation by cAMP-dependent kinase has minimal effect on this interaction, while phosphorylation by brain extracts or by purified calcium- and calmodulin-dependent kinase II reduces its actin-bundling and -binding activity. Synapsin's microtubule-binding activity, conversely, is stimulated after phosphorylation by the brain extract. Two complementary peptide fragments of synapsin generated by 2-nitro-5-thiocyanobenzoic cleavage and which map to opposite ends of the molecule participate in the bundling process, either by binding directly to actin or by binding to other synapsin I molecules. 2-Nitro-5-thiocyanobenzoic peptides arising from the central portion of the molecule demonstrate neither activity. In vivo, synapsin I may link small synaptic vesicles to the actin-based cortical cytoskeleton, and coordinate their availability for release in a Ca++-dependent fashion.

scholarly journals In vivo activity of glutaminase in the brain of hyperammonaemic rats measured by 15N nuclear magnetic resonance

1995 ◽  
Vol 305 (1) ◽  
pp. 329-336 ◽  
Author(s):  
K Kanamori ◽  
B D Ross
Keyword(s):  
Brain Homogenate ◽  
Inhibitory Effect ◽  
Intact Brain ◽  
Strong Inhibitory Effect ◽  
Glutamine Synthesis ◽  
Gaba Synthesis ◽  
The Brain

The in vivo activity of phosphate-activated glutaminase (PAG) was measured in the brain of hyperammonaemic rat by 15N n.m.r. Brain glutamine was 15N-enriched by intravenous infusion of 15NH4+ until the concentration of [5-15N]glutamine reached 6.1 mumol/g. Further glutamine synthesis was inhibited by intraperitoneal injection of methionine-DL-sulphoximine, an inhibitor of glutamine synthetase, and the infusate was changed to 14NH4+ during observation of decrease in brain [5-15N]glutamine due to PAG and other glutamine utilization pathways. Progressive decrease in brain [5-15N]glutamine, PAG-catalysed production of 15NH4+ and its subsequent assimilation into glutamate by glutamate dehydrogenase were monitored in vivo by 15N n.m.r. Brain [5-15N]glutamine (15N enrichment of 0.35-0.50) decreased at a rate of 1.2 mumol/h per g of brain. The in vivo PAG activity, determined from the observed rate and the quantity of 15NH4+ produced and subsequently assimilated into glutamate and aspartate, was 0.9-1.3 mumol/h per g. This activity is less than 1.1% of the reported activity in vitro measured in rat brain homogenate at a 10 mM concentration of the activator Pi. Inhibition by ammonia (brain level 1.4 mumol/g) alone does not account for the observed low activity in vivo. The result strongly suggests that, in intact brain, PAG activity is maintained at a low level by a suboptimal in situ concentration of Pi and the strong inhibitory effect of glutamate. The observed PAG activity in vivo is lower than the reported in vivo activity of glutamate decarboxylase which converts glutamate into gamma-aminobutyrate (GABA). The result suggests that PAG-catalysed hydrolysis of glutamine is not the sole provider of glutamate used for GABA synthesis.

scholarly journals Ribosomes in RNA granules are stalled on mRNA sequences that are consensus sites for FMRP association

2021 ◽  
Author(s):  
Mina N. Anadolu ◽  
Senthilkumar Kailasam ◽  
Konstanze Simbriger ◽  
Jingyu Sun ◽  
Teodora Markova ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Ribosome Profiling ◽  
Cytoskeletal Proteins ◽  
Rna Granules ◽  
Brain Extracts ◽  
The Brain

AbstractLocal translation in neurons is mediated in part by the reactivation of stalled polysomes. However, the mechanism for stalling of the polysomes is not understood. Stalled polysomes may be enriched within neuronal RNA granules defined by dense collections of compacted ribosomes found in the pellet of sucrose gradients used to separate polysomes from monosomes. We find that this fraction, isolated from P5 rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homolog (UPF1). Ribosome profiling of this fraction showed an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development and an enrichment of footprint reads on RNA binding proteins. Compared to those usually found in ribosome profiling studies, the footprint reads were more extended on their 3’end and were found in reproducible peaks in the mRNAs. These footprint reads were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the sedimented pellet to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall translation elongation in neurons, attracting FMRP and beginning a process where stalled ribosomes are packaged and transported in RNA granules.Significance StatementThis work finds that neuronal ribosomes in RNA granules are concentrated at consensus sites previously identified through cross-linking FMRP to mRNAs in the brain. This strongly links the compacted ribosomes found in the pellet of sucrose gradients from brain extracts to stalled ribosomes regulated by FMRP and provides important insights into how stalling is accomplished. Many mRNAs important for neurodevelopment are enriched in these ribosomes. These results suggest that many studies on translation in the brain may need to be revised. The larger size of the ribosomal footprints on stalled polysomes and their sedimentation in the pellet of sucrose gradients suggests mRNAs found in these structures have not been assessed in many studies of neuronal translation.

scholarly journals Proteomic Identification of Novel Substrates of a Protein Isoaspartyl Methyltransferase Repair Enzyme

2006 ◽  
Vol 281 (43) ◽  
pp. 32619-32629 ◽  
Author(s):  
Vasanthy Vigneswara ◽  
Jonathan D. Lowenson ◽  
Claire D. Powell ◽  
Matthew Thakur ◽  
Kevin Bailey ◽  
...  
Keyword(s):  
Repair Enzyme ◽  
Knock Out ◽  
Methyl Donor ◽  
Brain Extracts ◽  
Knock Out Mice ◽  
First Time ◽  
The Brain ◽  
Phosphatidylethanolamine Binding Protein ◽  
Mammalian Protein

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1-/-) accumulate more isoaspartyl residues than their wild-type littermates, with the most “damaged” residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1-/- mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-3H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1-/- animals compared with Pcmt1+/+ littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, β-synuclein, and α-synuclein, are all substrates for the l-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1-/- brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.

scholarly journals The Transport of Leucine and Aminocyclopentanecarboxylate across the Intact, Energy-Depleted Rat Blood—Brain Barrier

1989 ◽  
Vol 9 (2) ◽  
pp. 226-233 ◽  
Author(s):  
J. Greenwood ◽  
A. S. Hazell ◽  
O. E. Pratt
Keyword(s):  
Amino Acid ◽  
Blood Brain Barrier ◽  
Molecular Size ◽  
Brain Barrier ◽  
Large Neutral Amino Acid ◽  
Blood Brain ◽  
Order Of Magnitude ◽  
The Central Nervous System ◽  
The Brain

The transport across the blood-brain barrier of the large neutral amino acid leucine and the nonmetabolised aminocyclopentanecarboxylate (ACPC), of similar molecular size, was studied in the perfused, energy-depleted rat brain. It was found that when both leucine and ACPC were perfused for periods of up to 10 min their accumulation in the brain increased in a linear fashion. The ratio of perfusate radioactivity per milliliter and tissue radioactivity per gram (Rt/Rp) rose to above unity for both leucine and ACPC, indicating continued uptake against a concentration gradient of the radiolabel within the CNS. When the effect of increasing the concentration of the amino acid upon its influx into the brain was studied, it was found that under these conditions the kinetics of transport for both leucine and ACPC were of a similar order of magnitude to those reported previously in vivo. The values for the Michaelis constant for transport ( Km), maximum rate of transport ( Vmax), and the constant for the apparently linear, nonsaturable component ( Kd) for leucine into the cerebrum were 84.5 ± 29.0 μ M, 45.5 ± 1.5 nmole/min/g, and 2.62 ± 0.15 μl/min/g, respectively, and for ACPC 381 ± 64 μ M, 54.0 ± 1.5 nmole/min/g and 0.35 ± 0.10 μl/min/g, respectively. Comparing this data with previously reported values it is suggested that the transport of leucine into the central nervous system from a perfusate or bolus where no other competing amino acids are present, is flow dependent. Furthermore, ACPC enters the brain almost entirely by a carrier-mediated process, with little or no nonsaturable influx despite a similar oil/water partition coefficient as leucine.

scholarly journals N368-Tau fragments generated by legumain are detected only in trace amount in the insoluble Tau aggregates isolated from AD brain

2019 ◽  
Vol 7 (1) ◽  
Author(s):  
Kerstin Schlegel ◽  
Khader Awwad ◽  
Roland G. Heym ◽  
David Holzinger ◽  
Annika Doell ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Minor Component ◽  
Cleavage Sites ◽  
Brain Extracts ◽  
And Control ◽  
Progressive Accumulation ◽  
The Brain ◽  
Tissue Fractionation

Abstract Intraneuronal insoluble inclusions made of Tau protein are neuropathological hallmarks of Alzheimer Disease (AD). Cleavage of Tau by legumain (LGMN) has been proposed to be crucial for aggregation of Tau into fibrils. However, it remains unclear if LGMN-cleaved Tau fragments accumulate in AD Tau inclusions. Using an in vitro enzymatic assay and non-targeted mass spectrometry, we identified four putative LGMN cleavage sites at Tau residues N167-, N255-, N296- and N368. Cleavage at N368 generates variously sized N368-Tau fragments that are aggregation prone in the Thioflavin T assay in vitro. N368-cleaved Tau is not detected in the brain of legumain knockout mice, indicating that LGMN is required for Tau cleavage in the mouse brain in vivo. Using a targeted mass spectrometry method in combination with tissue fractionation and biochemical analysis, we investigated whether N368-cleaved Tau is differentially produced and aggregated in brain of AD patients and control subjects. In brain soluble extracts, despite reduced uncleaved Tau in AD, levels of N368-cleaved Tau are comparable in AD and control hippocampus, suggesting that LGMN-mediated cleavage of Tau is not altered in AD. Consistently, levels of activated, cleaved LGMN are also similar in AD and control brain extracts. To assess the potential accumulation of N368-cleaved Tau in insoluble Tau aggregates, we analyzed sarkosyl-insoluble extracts from AD and control hippocampus. Both N368-cleaved Tau and uncleaved Tau were significantly increased in AD as a consequence of pathological Tau inclusions accumulation. However, the amount of N368-cleaved Tau represented only a very minor component (< 0.1%) of insoluble Tau. Our data indicate that LGMN physiologically cleaves Tau in the mouse and human brain generating N368-cleaved Tau fragments, which remain largely soluble and are present only in low proportion in Tau insoluble aggregates compared to uncleaved Tau. This suggests that LGMN-cleaved Tau has limited role in the progressive accumulation of Tau inclusions in AD.

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