Association of botulinum neurotoxin serotype A light chain with plasma membrane-bound SNAP-25

The Clostridium botulinum neurotoxins (BoNTs) cleave SNARE proteins, which inhibit binding and thus fusion of neurotransmitter vesicles to the plasma membrane of peripheral neurons. BoNTs comprise an N-terminal light chain (LC) and C-terminal heavy chain, which are linked by a disulfide bond. There are seven serotypes (A-G) of BoNTs based upon immunological neutralization. Although the binding and entry of BoNT/A into neurons has been subjected to considerable investigation, the intracellular events that allow BoNT/A to efficiently cleave SNAP-25 within neurons is less well understood. Earlier studies showed that intracellular LC/A bound to the plasma membrane of neurons. In this study, intracellular LC/A is shown to directly bind SNAP-25 on the plasma membrane. Solid phase binding showed that the N-terminal residues of LC/A bound residues 80-110 of SNAP-25, which was also observed in cultured neurons. Association of the N-terminal 8 amino acids of LC/A and residues 80-110 of SNAP-25 also enhanced substrate cleavage. These findings explain how LC/A associates with SNAP-25 on the plasma membrane and provide a basis for LC/A cleavage of SNAP-25 within the SNARE complex.     

Surface properties of fibrinogen and fibrin

By contact angle measurements on layers of fibrinogen and fibrin, it can be shown that the transformation from fibrinogen to fibrin is accompanied by a change in surface properties from very hydrophilic (fibrinogen) to moderately but definitely hydrophobic (fibrin). It is also shown that, contrary to serum albumin and gamma globulin, fibrinogen does not become more hydrophobic upon drying.     

献血员抗－HCV检测报告

对１５８７名献血员进行了抗－ＨＣＶ检测,总阳性率５．６０％,在不同职业中农民较高,占６．７２％,３０岁以上为１２．７６％。我站自改进采浆工艺,严格使用一次性消毒器具后,未发现ＨＣＶ交叉感染。职业献血者抗－ＨＣＶ阳性率高达３５．４８％,易造成输血后ＨＣＶ感染。对ＨＣＶ感染ＯＤ值阳性３０人又进行４－６个月的观察复测,其中１６人ＯＤ值下降,６人上升,认为ＯＤ值的变化与自限性ＨＣＶ感染有关。     

Purification and partial characterisation of α2-antiplasmin and plasmin(ogen) from ostrich plasma

This study reports the isolation and partial characterisation of the ostrich serpin, α₂AP, and its target enzyme, ostrich plasmin, in its active and inactive proenzyme, namely plasminogen, forms. Ostrich α₂AP was purified using lysine–Sepharose chromatography, ammonium sulfate fractionation, and Super Q-650S and ostrich LBSI–Sepharose chromatographies. It revealed a M_r of 84 K (thousand) and had one and two N-terminal amino acids in common with 11 of those of human and bovine α₂AP, respectively. It showed the largest inhibitory effect on ostrich plasmin, followed by bovine trypsin and plasmin, respectively, and much less plasmin inhibition than bovine aprotinin, but much more so than human α₂AP, DFP and EACA. Ostrich plasminogen was highly purified after lysine–Sepharose chromatography and showed a M_r of 92 K, a total of 775 amino acids and its N-terminal sequence showed 53% identity with those of human, rabbit, cat, and ox plasminogens. Ostrich plasmin, obtained by the urokinase-activation of ostrich plasminogen, revealed a M_r of 78 K, a total of 638 amino acids, an N-terminal sequence showing two to four residues identical to five of those of human, cat, dog, rabbit, and ox plasmins, and pH and temperature optima of 8.0 and 40°C, respectively.     

pH-dependent cargo sorting from the Golgi

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.     

Responses of plasma human atrial natriuretic factor to high intensity submaximal exercise in the heat

No data exists regarding responses of human atrial natriuretic factor (ANF) to exercise in the heat. The purpose of this study was to examine the responses of plasma ANF to high intensity submaximal (71% +/- 0.9 VO2max) exercise in the heat over an eight day acclimation period. Fourteen healthy males volunteered to participate in the study. Subjects performed intermittent exercises on a treadmill (0% grade) during 50 min of each 100 min trial in an environmental chamber maintained at 41.2 +/- 0.5 degrees C, 39.0 +/- 1.7% relative humidity. Blood was obtained from an antecubital vein after standing 20 min in the heat prior to exercise, and immediately after exercise. Measures were compared on days 1, 4 and 8. ANF did not change pre- to post-exercise nor did it change over the eight day heat acclimation period despite other heat acclimation adaptations. Conversely, plasma aldosterone (ALDO), renin activity (PRA) and cortisol (COR) all increased (p less than 0.05) pre- to post-exercise on each day but again no changes were observed over the eight day period. These data support that ANF may not increase when ALDO and PRA increases are observed.     

Permeation through Phospholipid Bilayers,Skin‐Cell Penetration,Plasma Stability,and CD Spectra of α‐ and β‐Oligoproline Derivatives

After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell‐penetrating properties of polycationic proline‐containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002 , 124, 8876) is acknowledged, according to which fluorescein‐labeled tetradecaproline is slowly taken up by rat kidney cells (NRK‐49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004 , 1, 1111) observation that a hexa‐β³‐Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo‐L ‐ and oligo‐D ‐α‐prolines, as well as of oligo‐β²h‐ and oligo‐β³h‐prolines without and with fluorescence labels ( 1 – 8 ; Fig. 1). Permeation through protein‐free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2–4). This methodology is applied for the first time for quantitative determination of translocation rates of cell‐penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6–8, resp.). The stabilities of oligoprolines in heparin‐stabilized human plasma increase with decreasing chain lengths (Figs. 9–11). Time‐ and solvent‐dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.     

Plasmalemma-associated malate dehydrogenase activity in onion root cells

Summary Plasma membrane vesicles isolated from onion roots showed oxaloacetate reductase activity as well as other oxidoreductase activities. Purification and further sequencing showed that the protein responsible for the activity is a 40 kDa protein which corresponds to the cytosolic soluble malate dehydrogenase. However, the activity remained bound to the membrane after repeated freezing and thawing cycles and further washing, excluding a cytosolic contamination as the source of the activity. Furthermore, a second 28 kDa protein has been copurified together with the 40 kDa protein. The plasmalemma oxaloacetate reductase activity shows both donor and acceptor sites located towards the cytoplasmic side of the plasma membrane. This enzyme catalyzed the oxidation of NADH by oxaloacetate and the reduction of NAD⁺ by malate in the presence of an oxaloacetate-withdrawing system. We conclude that a significant amount of the cytosolic malate dehydrogenase can be specifically attached to the cytosolic face of the plasmalemma. A possible role in a putative malate shuttle associated to the plasma membrane is discussed.Abbreviations AFR ascorbate free radical - DQ duroquinone - OA oxaloacetate - DPIP dichlorophenolindophenol - MDH malate dehydrogenase - PHMB p-hydroxymercuribenzoate     

L-Citrulline acts as potential hypothermic agent to afford thermotolerance in chicks

Recently we demonstrated that L-citrulline (L-Cit) causes hypothermia in chicks. However, the question of how L-Cit mediates hypothermia remained elusive. Thus, the objective of this study was to examine some possible factors in the process of L-Cit-mediated hypothermia and to confirm whether L-Cit can also afford thermotolerance in young chicks. Chicks were subjected to oral administration of L-Cit along with intraperitoneal injection of a nitric oxide synthase (NOS) inhibitor, N^G-nitro-l-arginine methyl ester HCl (L-NAME), to examine the involvement of NO in the process of hypothermia. Food intake and plasma metabolites were also analyzed after oral administration of L-Cit in chicks. To examine thermotolerance, chicks were orally administered with a single dose of L-Cit (15 mmol/10 ml/kg body weight) or the same dose twice within a short interval of 1 h (dual oral administration) before the exposure to high ambient temperature (35 ± 1 °C) for 180 min. Although the rectal temperature was reduced following administration of L-Cit, L-NAME caused a greater reduction. L-NAME reduced total NO₂ and NO₃ (NOx) in plasma, which confirmed its inhibitory effect on NO. A single oral administration of L-Cit mediated a persistent state of hypothermia for the 300 min of the study without affecting food intake. It was further found that plasma glucose was significantly lower in L-Cit-treated chicks. Dual oral administration of L-Cit, but not a single oral administration, afforded thermotolerance without a significant change in plasma NOx in chicks. In conclusion, our results suggest that L-Cit-mediated hypothermia and thermotolerance may not be involved in NO production. L-Cit-mediated thermotolerance further suggests that L-Cit may serve as an important nutritional supplement that could help in coping with summer heat.     

Comparison of the properties of active Ca2+ transport by the islet-cell endoplasmic reticulum and plasma membrane

The properties of active or ATP-dependent calcium transport by islet-cell endoplasmic reticulum and plasma membrane-enriched subcellular fractions were directly compared. These studies indicate that the active calcium transport systems of the two membranes are fundamentally distinct. In contrast to calcium uptake by the endoplasmic reticulum-enriched fraction, calcium uptake by islet-cell plasma membrane-enriched vesicles exhibited a different pH optimum, was not sustained by oxalate, and showed an approximate 30-fold greater affinity for ionized calcium. A similar difference in affinity for calcium was exhibited by the Ca²⁺-stimulated ATPase activities which are associated with these islet-cell subcellular fractions. Consistent with the effects of calmodulin on calcium transport, calmodulin stimulated Ca²⁺-ATPase in the plasma membranes, but did not increase calcium-stimulated ATPase activity in the endoplasmic reticulum membranes. The physiological significance of the differences observed in calcium transport by the endoplasmic reticulum and plasma membrane fractions relative to the regulation of insulin secretion by the islets of Langerhans is discussed.