Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation

A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of¹⁴C-glutamine to¹⁴CO₂ and of¹⁴C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle -ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label -ketoglutarate carbon 5, [2-¹⁴C]glucose, [1-¹⁴C]acetate and [5-¹⁴C]glutamine. The analysis indicated that the probability of flux of TCA cycle -ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of -ketoglutarate through -ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [¹⁴C]glutamine to¹⁴CO₂, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through -ketoglutarate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of -ketoglutarate was demonstrated by measuring incorporation of [5-¹⁴C]glutamine into¹⁴C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells.     

Selective dimerization of cysteines in glycopeptides and phosphopeptides

Summary To study the influence of phosphorylation and oxidation on the repeat domains of human Tau protein, we faced the challenge to selectively dimerize two cysteine-containing peptides in the presence of a nearby phosphate group. To this end, we were able to demonstrate the utility of a selective dimerization approach by forming disulfide bonds in unprotected phosphopeptides and extended the methodology to unprotected glycopeptides. Activation of one cysteine of a peptide chain with 2,2-dithiodipyridine and coupling this thiopyridyl-peptide to another peptide chain, containing an unprotected cysteine residue, yielded the mixed dimers in high purities and reasonable yields. Phosphate or sugar side chains on either peptide component remained unaffected during the activation and dimerization processes. While for mixed dimers the activated peptides were isolated by chromatography, homodimers were obtained by a simple one-pot reaction after 1 h. We demonstrate that cysteines can be dimerized in unprotected phosphopeptides and glycopeptides, without any side reactions affecting these posttranslational modifications.Abbreviations DCM dichloromethane - DMF N,N-dimethylformamide - DTP 2,2-dithiodipyridine - Fmoc 9-fluorenylmethyloxycarbonyl - HPLC high-performance liquid chromatography - MALDI matrix-assisted laser desorption/ionization - MS mass spectrometry - NFT neurofibrillary tangles - PHF paired helical filaments - PKC protein kinase C - RP reversed phase - human Tau protein - TFA trifluoroacetic acid Parts of this paper were presented at the 24th European Peptide Symposium in Edinburgh, Scotland, U.K., September 8–13, 1996.     

Tau Released from Paired Helical Filaments with Formic Acid or Guanidine Is Susceptible to Calpain-Mediated Proteolysis

Abstract: Paired helical filaments (PHFs), a characteristic neuropathologic finding in Alzheimer's disease brain, are abnormal fibrillary forms of hyperphosphorylated tau (PHF-tau), which have been shown to be highly resistant to calpain digestion. Either excessive phosphorylation or fibrillary arrangement of tau proteins in PHFs may play a role in proteolytic resistance by limiting access to calpain recognition/digestion sites. To determine the contribution of the fibrillary conformation, isolated PHFs were subjected to treatment with either formic acid or guanidine. Both procedures effectively abolished the fibrillary structure of PHF but preserved PHF-tau immunoreactivity using a panel of antibodies that recognize nonphosphorylated and phosphorylated epitopes. These treatments also significantly increased the sensitivity of PHF-tau polypeptides to calpain proteolysis as shown by significant decreases in the half-life ( t _1/2) from the infinite with native PHF to 44 min and 4.4 min in formic acid- or guanidine-treated samples, respectively. In contrast, the sensitivity of normal fetal tau (3.4 min) was either decreased (5.9 min) or unaffected (3.6 min) by similar treatment. Our results indicate that after guanidine treatment, the sensitivity of PHF to calpain resembles that of fetal tau. These results strongly suggest that the fibrillary structure of PHF-tau, rather than hyperphosphorylation, is the major factor responsible for the resistance of abnormal filaments to calpain-mediated proteolysis.     

Vascular permeability to proteins and peptides in the mouse pineal gland

Summary The permeability of fenestrated capillaries in the mouse pineal gland to proteins and peptides was demonstrated by means of ultrastructural tracers. Horseradish peroxidase (HRP) and microperoxidase (MP) were injected intravenously and allowed to circulate for approximately 30 s, 1 min, 5 min, 1 or 2h. The tissue was then fixed by vascular perfusion or by immersion with aldehydes. In all experiments a pronounced extravasation of HRP and MP occurred. Transendothelial vesicular transport seemed to have occurred across the fenestrated capillaries. The most pronounced tracer labeling of vesicles was found after 1 min of MP- or HRP-circulation. The vesicles were uncoated and more than 70 % of the HRP-and MP-containing vesicles exhibited diameters between 50 and 110 nm. Furthermore, three other transcapillary pathways taken by the tracers are suggested: 1) via intercellular junctions, 2) through fenestrae and 3) via channels formed by fusion of vesicles with the luminal and abluminal cell membranes. Based on these results, it is assumed that the capillaries in the mouse pineal gland are also permeable to peptides synthesized and secreted by the pineal gland.Part of this study was presented at the EMCELL-76 meeting, Copenhagen, 1976     

Observations of the pattern of secondary wall development in the hypodermis of onion (Allium cepa) roots

Summary This brief communication reports the appearance, under certain circumstances, of root hypodermal cells with transfer cell labyrinths. These cells lie at regular intervals around the hypodermis at the bases of onion bulb roots. They are narrower (smaller tangential dimension) than unmodified cells but have the same radial dimension. These narrow cells contain small vacuoles, their main volume being composed of a cytoplasm rich in organelles, especially mitochondria. When treated with a low concentration of lanthanum nitrate solution, the tracer accumulates in the outer tangential wall and in small vacuoles and vesicles.     

Characterization of a pilot plant airlift tower loop bioreactor: II. Evaluation of global mixing properties of the gas phase during yeast cultivation

Saccharomyces cerevisiae was cultivated in a 4-m(3) pilot plant airlift tower loop reactor with a draft tube in batch and continuous operations and for comparison in a laboratory airlift tower loop reactor of 0.08 m(3) volume. The reactors were characterized during and after the cultivation by measuring the distributions of the residence times of the gas phase with pseudostochastic tracer signals and mass spectrometer and by evaluating the mixing in the liquid phase with a pulse-shaped volatile tracer signal and mass spectrometer as a detector. The mean residence times and the intensities of the axial mixing in the riser and downcomer, the circulation times of the gas phase, and the fraction of the recirculated gas phase were evaluated and compared.     

Tau inhibits PKA by nuclear proteasome‐dependent PKAR2α elevation with suppressed CREB/GluA1 phosphorylation

Intraneuronal accumulation of wild‐type tau plays a key role in Alzheimer's disease, while the mechanisms underlying tauopathy and memory impairment remain unclear. Here, we report that overexpressing full‐length wild‐type human tau (hTau) in mouse hippocampus induces learning and memory deficits with remarkably reduced levels of multiple synapse‐ and memory‐associated proteins. Overexpressing hTau inhibits the activity of protein kinase A (PKA) and decreases the phosphorylation level of cAMP‐response element binding protein (CREB), GluA1, and TrkB with reduced BDNF mRNA and protein levels both in vitro and in vivo. Simultaneously, overexpressing hTau increased PKAR2α (an inhibitory subunit of PKA) in nuclear fraction and inactivated proteasome activity. With an increased association of PKAR2α with PA28γ (a nuclear proteasome activator), the formation of PA28γ‐20S proteasome complex remarkably decreased in the nuclear fraction, followed by a reduced interaction of PKAR2α with 20S proteasome. Both downregulating PKAR2α by shRNA and upregulating proteasome by expressing PA28γ rescued hTau‐induced PKA inhibition and CREB dephosphorylation, and upregulating PKA improved hTau‐induced cognitive deficits in mice. Together, these data reveal that intracellular tau accumulation induces synapse and memory impairments by inhibiting PKA/CREB/BDNF/TrkB and PKA/GluA1 signaling, and deficit of PA28γ‐20S proteasome complex formation contributes to PKAR2α elevation and PKA inhibition.     

Discovery of biphenyl pyrazole scaffold for neurodegenerative diseases: A novel class of acetylcholinesterase-centered multitargeted ligands

Multitargeted ligands have demonstrated remarkable efficiency as potential therapeutics for neurodegenerative diseases as they target multiple pathways involved in the progression of these diseases. Herein, we report first-in-class dual inhibitor of acetylcholinesterase (AChE) and tau aggregation as a novel class of multitargeted ligands for neurodegenerative diseases. The reported biphenyl pyrazole scaffold binds monomeric tau with submicromolar affinity and impedes the formation of tau oligomers at early stages. Additionally, the lead compound inhibited AChE activity with an IC₅₀ value of 0.35 ± 0.02 μM. Remarkably, the neuroprotective effect of this lead in induced cytotoxicity model of SH-SY5Y neuroblastoma cells is superior to single-targeted AChE and tau-aggregation inhibitors. This scaffold would enable development of new generation of multitargeted ligands for neurodegenerative diseases that function through dual targeting of AChE and monomeric tau.