Measurement of endogenous leucine enkephalin in canine caudate nuclei and hypothalami with high-performance liquid chromatography and field-desorption mass spectrometry

For the first time, endogenous amounts of Leu-enkephalin are measured in brain tissue with a technique preserving integrity of the entire molecular structure of the neuropeptide. Field-desorption mass spectrometry enables measurement of picomole amounts of endogenous, chemically underivatized Leu-enkephalin in canine caudate nuclei and hypothalami. The optimal sensitivity and resolution of high-performance liquid chromatography is coupled with maximal molecular specificity of field-desorption mass spectrometry to measure enkephalins in caudate nuclei and hypothalami from dog brains. This novel combination of two recent instrumental methodologies provides a firm molecular basis for calibrating the radioimmunoassay measurement of endogenous levels of biologically active brain neuropeptides.     

Hepatic metabolism of prostacyclin (PGI2) in the rabbit: Formation of a potent novel inhibitor of platelet aggregation

Metabolism of [9-³H]-PGI₂ was studied in the isolated Tyrode's perfused rabbit liver. Five products, four radioactive and one non-radioactive, were identified in the perfusate: 19-hydroxy-6-keto-PGF_1α, 6-keto-PGF_1α, dinor-6-keto-PGF_1α, pentanor PGF_1α and a 6-keto-PGE₁-like substance. The first two, 19-hydroxy-6-keto-PGF_1α and 6-keto-PGF_1α, represented 5% and 45% respectively, of the total radioactivity; the last two accounted for 39%. The presence of dinor and pentanor derivatives of 6-keto-PGF_1α indicated that β -oxidation and oxidative-decarboxylation occurs in the liver as the major metabolic pathway of PGI₂. One non-radioactive metabolite which co-migrated with authentic 6-keto-PGE₁ was found to inhibit platelet aggregation, having a potency similar to authentic 6-keto-PGE₁, and its effect can be eliminated by boiling and by alkali treatment. This metabolite, having similar Rf value on TLC and biological behavior as 6-keto-PGE₁, may arise from oxidation of 6-keto-PGF_1α via the 9-hydroxyprostaglandin dehydrogenase pathway, as suggested by recovery of tritiated water in the aqueous phase of the perfusate. This material, a potent inhibitor of platelet aggregation, may arise from PGI₂ or its hydrolysis product, 6-keto-PGF_1α.     

Mass spectrometry of acetylated,permethylated and reduced oligopeptides

The amino acid sequence of oligopeptides can be determined from the mass spectra of their volatile derivatives. New peptide derivatives are described which are prepared from permethylated peptides by reduction with borane-tetrahydrofuran. These derivatives have been found to be more volatile than the corresponding permethylated ones. The procedure is illustrated by application of this technique to representative oligopeptides. As little as 10–100 nmol of these peptides have been sequenced using this technique.     

Na+/K+-ATPase is present in scrapie-associated fibrils, modulates PrP misfolding in vitro and links PrP function and dysfunction

Transmissible spongiform encephalopathies are characterised by widespread deposition of fibrillar and/or plaque-like forms of the prion protein. These aggregated forms are produced by misfolding of the normal prion protein, PrP(C), to the disease-associated form, PrP(Sc), through mechanisms that remain elusive but which require either direct or indirect interaction between PrP(C) and PrP(Sc) isoforms. A wealth of evidence implicates other non-PrP molecules as active participants in the misfolding process, to catalyse and direct the conformational conversion of PrP(C) or to provide a scaffold ensuring correct alignment of PrP(C) and PrP(Sc) during conversion. Such molecules may be specific to different scrapie strains to facilitate differential prion protein misfolding. Since molecular cofactors may become integrated into the growing protein fibril during prion conversion, we have investigated the proteins contained in prion disease-specific deposits by shotgun proteomics of scrapie-associated fibrils (SAF) from mice infected with 3 different strains of mouse-passaged scrapie. Concomitant use of negative control preparations allowed us to identify and discount proteins that are enriched non-specifically by the SAF isolation protocol. We found several proteins that co-purified specifically with SAF from infected brains but none of these were reproducibly and demonstrably specific for particular scrapie strains. The α-chain of Na(+)/K(+)-ATPase was common to SAF from all 3 strains and we tested the ability of this protein to modulate in vitro misfolding of recombinant PrP. Na(+)/K(+)-ATPase enhanced the efficiency of disease-specific conversion of recombinant PrP suggesting that it may act as a molecular cofactor. Consistent with previous results, the same protein inhibited fibrillisation kinetics of recombinant PrP. Since functional interactions between PrP(C) and Na(+)/K(+)-ATPase have previously been reported in astrocytes, our data highlight this molecule as a key link between PrP function, dysfunction and misfolding.     

Exosomes surf on filopodia to enter cells at endocytic hot spots,traffic within endosomes,and are targeted to the ER

Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.     

β-Amyloid carrying the Dutch mutation has diverse effects on calpain-mediated toxicity in hippocampal neurons

Hereditary cerebral hemorrhage with amyloidosis-Dutch type is a disorder associated with a missense mutation (E693Q) in the β-amyloid (Aβ)-coding region of the amyloid precursor protein (APP). This familial disease is characterized by cognitive deficits secondary to intracerebral hemorrhage and, in some cases, progressive Alzheimer's disease (AD)-like dementia. Although this mutation was the first ever reported in the human APP gene, little is known about the molecular mechanisms underlying the direct toxic effects of this mutated Aβ on central neurons. In the present study, we assessed the role of calpain-mediated toxicity in such effects using an AD primary culture model system. Our results showed that Dutch mutant Aβ (E22Q) induced calpain-mediated cleavage of dynamin 1 and a significant decrease in synaptic contacts in mature hippocampal cultures. These synaptic deficits were similar to those induced by wild-type (WT) Aβ. In contrast, calpain-mediated tau cleavage leading to the generation of a 17-kDa neurotoxic fragment, as well as neuronal death, were significantly reduced in E22Q Aβ-treated neurons when compared with WT Aβ-treated ones. This complex regulation of the calpain-mediated toxicity pathway by E22Q Aβ could have some bearing in the pathobiology of this familial AD form.     

Chlamydomonas raudensis (UWO 241), Chlorophyceae,exhibits the capacity for rapid D1 repair in response to chronic photoinhibition at low temperature1

Maximum photosynthetic capacity indicates that the Antarctic psychrophile Chlamydomonas raudensis H. Ettl UWO 241 is photosynthetically adapted to low temperature. Despite this finding, C. raudensis UWO 241 exhibited greater sensitivity to low‐temperature photoinhibition of PSII than the mesophile Chlamydomonas reinhardtii P. A. Dang. However, in contrast with results for C. reinhardtii, the quantum requirement to induce 50% photoinhibition of PSII in C. raudensis UWO 241 (50 μmol photons) was comparable at either 8°C or 29°C. To our knowledge, this is the first report of a photoautotroph whose susceptibility to photoinhibition is temperature independent. In contrast, the capacity of the psychrophile to recover from photoinhibition of PSII was sensitive to temperature and inhibited at 29°C. The maximum rate of recovery from photoinhibition of the psychrophile at 8°C was comparable to the maximum rate of recovery of the mesophile at 29°C. We provide evidence that photoinhibition in C. raudensis UWO 241 is chronic rather than dynamic. The photoinhibition‐induced decrease in the D1 content in C. raudensis recovered within 30 min at 8°C. Both the recovery of the D1 content as well as the initial fast phase of the recovery of F_v/F_m at 8°C were inhibited by lincomycin, a chloroplast protein synthesis inhibitor. We conclude that the susceptibility of C. raudensis UWO 241 to low‐temperature photoinhibition reflects its adaptation to low growth irradiance, whereas the unusually rapid rate of recovery at low temperature exhibited by this psychrophile is due to a novel D1 repair cycle that is adapted to and is maximally operative at low temperature.     

Carbon isotope evidence for recent climate‐related enhancement of CO 2 assimilation and peat accumulation rates in Antarctica

Signy Island, maritime Antarctic, lies within the region of the Southern Hemisphere that is currently experiencing the most rapid rates of environmental change. In this study, peat cores up to 2 m in depth from four moss banks on Signy Island were used to reconstruct changes in moss growth and climatic characteristics over the late Holocene. Measurements included radiocarbon dating (to determine peat accumulation rates) and stable carbon isotope composition of moss cellulose (to estimate photosynthetic limitation by CO ₂ supply and model CO ₂ assimilation rate). For at least one intensively ¹⁴C‐dated Chorisodontium aciphyllum moss peat bank, the vertical accumulation rate of peat was 3.9 mm yr^?1 over the last 30 years. Before the industrial revolution, rates of peat accumulation in all cores were much lower, at around 0.6–1 mm yr^?1. Carbon‐13 discrimination (Δ), corrected for background and anthropogenic source inputs, was used to develop a predictive model for CO ₂ assimilation. Between 1680 and 1900, there had been a gradual increase in Δ, and hence assimilation rate. Since 1800, assimilation has also been stimulated by the changes in atmospheric CO ₂ concentration, but a recent decline in Δ (over the past 50–100 years) can perhaps be attributed to documented changes in temperature and/or precipitation. The overall increase in CO ₂ assimilation rate (¹³C proxy) and enhanced C accumulation (¹⁴C proxy) are consistent with warmer and wetter conditions currently generating higher growth rates than at any time in the past three millennia, with the decline in Δ perhaps compensated by a longer growing season.