Identifying regions of membrane proteins in contact with phospholipid head groups: covalent attachment of a new class of aldehyde lipid labels to cytochrome c oxidase

A series of amine-specific reagents based on the benzaldehyde reactive group have been synthesized, characterized, and used to study beef heart cytochrome c oxidase reconstituted in phospholipid bilayers. The series contained three classes of reagents: lipid-soluble phosphodiesters having a single hydrocarbon chain, phospholipid analogues, and a water-soluble benzaldehyde. All reagents were either radiolabeled or spin-labeled or both. The Schiff bases formed by these benzaldehydes with amines were found to be reversible until the addition of the reducing agent sodium cyanoborohydride, whereas attachment of lipid-derived aliphatic aldehydes was not readily reversible in the absence of the reducing agent. The benzaldehyde group provides a convenient method of controlling and delaying permanent attachment to integral membrane proteins until after the reconstitution steps. This ensures that the lipid analogues are located properly to identify amine groups at the lipid-protein interface rather than reacting indiscriminately with amines of the hydrophilic domains of the protein. The benzaldehyde lipid labels attach to cytochrome c oxidase with high efficiency. Typically, 20% of the amount of lipid label present was covalently attached to the protein, and the number of moles of label incorporated per mole of protein ranged from 1 to 6, depending on the molar ratios of label, lipid, and protein. The efficiency of labeling by the water-soluble benzaldehyde was much less than that observed for any of the lipid labels because of dilution effects, but equivalent levels of incorporation were achieved by increasing the label concentration. Electron spin resonance spectra of a nitroxide-containing phospholipid analogue covalently attached to reconstituted cytochrome c oxidase exhibited a large motion-restricted component, which is characteristic of spin-labeled lipids in contact with the hydrophobic surfaces of membrane proteins. The line shape and splittings were similar for covalently attached label and label free to diffuse and contact the protein molecules in the bilayer, providing independent evidence that the coupling occurs at the protein-lipid interface. The distribution of the benzaldehyde reagents attached to the polypeptide components of cytochrome c oxidase was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeling pattern observed for the lipid analogues was not affected by the presence of the nitroxide moiety on the acyl chains but was dependent on the molar ratio of labeling reagent to protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protoplast fusion ofCatharanthus roseus cells by electrofusion of chemically-agglutinated protoplasts

Mesophyll derived protoplasts ofCatharanthus roseus cv. Little pinkie were fused with protoplasts derived from an habituated cell line ofC. roseus. Polyethylene glycol was used as agglutinating agent while fusions were induced by square pulses. Best results were obtained by fusing protoplasts from primary leaves with those from three-day-old cell cultures. Adding calcium ions considerably enhanced heterofusion rate. Good cell viabilities indicated that this fusion process was not cytotoxic. The heterofusion frequency was up to 10% or more. Most of the heterokaryons were able to regenerate their cell walls and underwent division. Communicated by J. TUPY     

Multiple feather follicle cysts in a wild turkey

A hunter-killed wild turkey (Meleagris gallopavo silvestris) was submitted for examination because of numerous 2 to 30 mm diameter, yellowish, hard nodules in the skin. The nodules were confined to the skin and did not involve subcutaneous tissues. Nodules consisted of dilated feather follicles packed with a caseous tan to pale yellow material. Histologically, affected feather follicles were markedly dilated and filled with laminated keratin debris. The lesions were determined to be multiple feather follicle cysts of unknown etiology.     

Changes in IL-6, IL-8, C-reactive protein and pancreatic secretory trypsin inhibitor after transcatheter arterial chemo-embolization therapy for hepato-cellular carcinoma.

In an attempt to investigate the interaction between the changes of cytokines and acute phase reactants after transcatheter arterial chemoembolization therapy (TACE), the levels of interleukin 6 (IL-6), interleukin 8 (IL-8), C-reactive protein (CRP) and pancreatic secretory trypsin inhibitor (PSTI) in the blood of patients with unresectable hepatocellular carcinoma (HCC) were measured. Before the therapy, serum IL-6 and plasma IL-8 levels were detectable in 77.8% and 28.5%, respectively, of patients with HCC. Levels of serum IL-6 and plasma IL-8 increased after TACE and reached a peak on day 3 in all patients (18/18) and in 87.5% of patients (12/14), respectively. Both blood levels of IL-6 and IL-8 reached a peak earlier than those of CRP and PSTI did after the therapy. When the maximal values of IL-6 were compared with those of CRP and PSTI, there were significant positive correlations (r = 0.63, P < 0.01 and r = 0.81, P < 0.01, respectively). Similarly, comparisons of the maximal values of IL-8 with those of CRP and PSTI gave a significant correlation (r = 0.68, P < 0.01 and r = 0.67, P < 0.05, respectively). However, no significant correlation was found between the elevation of IL-6 and IL-8.     

Lipase catalyzed reactions of aliphatic and arylaliphatic carbonic acid esters

Symmetrical dialkyl carbonates and dibenzyl carbonates reacted with various nucleophiles in the presence of Candida antarctica lipase B in organic solvents. For example, reaction of dibutyl and dibenzyl carbonate with an alcohol gave a mixture of the mono- and disubstituted products. Aminolysis, however, afforded only the carbamates, without subsequent reaction to the ureum derivatives. The reaction rates were rather low compared with carboxylic esters; the reactivity increased in the order dimethyl相似文献   

Chronic hypoxia attenuates resting and exercise-induced VEGF, flt-1, and flk-1 mRNA levels in skeletal muscle.

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic mitogen. However, chronic hypoxia is generally not found to increase mammalian skeletal muscle capillarity. We sought to determine the effect of chronic hypoxia (8 wk, inspired O2 fraction = 0.12) on skeletal muscle gene expression of VEGF, its receptors (flt-1 and flk-1), basic fibroblast growth factor, and transforming growth factor-beta1. Wistar rats were exposed to chronic hypoxia (n = 12) or room air (n = 12). After the exposure period, six animals from each group were subjected to a single 1-h treadmill exercise bout (18 m/min on a 10 degrees incline) in room air while the remaining six animals served as rest controls. Morphological analysis revealed that chronic hypoxia did not increase skeletal muscle capillarity. Northern blot analyses showed that chronic hypoxia decreased resting VEGF, flt-1, and flk-1 mRNA by 23, 68, and 42%, respectively (P < 0.05). The VEGF mRNA response to exercise was also decreased (4.1- and 2.7-fold increase in room air and chronic hypoxia, respectively, P < 0.05). In contrast, neither transforming growth factor-beta1 nor basic fibroblast growth factor mRNA was significantly altered by chronic hypoxia. In conclusion, prolonged exposure to hypoxia attenuated gene expression of VEGF and its receptors flt-1 and flk-1 in rat gastrocnemius muscle. These findings may provide an explanation for the lack of mammalian skeletal muscle angiogenesis that is observed after chronic hypoxia.     

Isolation and characterization of microsatellites in Pacific white shrimp Penaeus (Litopenaeus) vannamei

Five polymorphic microsatellite loci were characterized for Penaeus (Litopenaeus) vannamei. Loci were isolated using a partial Sau3A1 genomic library by the sequencing of randomly selected clones and by a biotinylated (CT)₁₀ and (GT)₁₀ probes screening procedure. The last strategy resulted in the most useful data. About 40% of the clones showed a previously reported satellite/microsatellite (PVS1), reducing the chance of finding new microsatellite regions. Whereas two of the microsatellite loci with more than 10 alleles will be useful for mating analysis in a breeding program, the others might prove useful for population genetic studies.     

In vivo neutralization of TNF-alpha promotes humoral autoimmunity by preventing the induction of CTL.

Neutralization of TNF-alpha in humans with rheumatoid arthritis or Crohn's disease has been associated with the development of humoral autoimmunity. To determine the effect of TNF-alpha neutralization on cell-mediated and humoral-mediated responses, we administered anti-TNF-alpha mAb to mice undergoing acute graft-vs-host disease (GVHD) using the parent-into-F(1) model. In vivo neutralization of TNF-alpha blocked the lymphocytopenic features characteristic of acute GVHD and induced a lupus-like chronic GVHD phenotype (lymphoproliferation and autoantibody production). These effects resulted from complete inhibition of detectable antihost CTL activity and required the presence of anti-TNF-alpha mAb for the first 4 days after parental cell transfer, indicating that TNF-alpha plays a critical role in the induction of CTL. Moreover, an in vivo blockade of TNF-alpha preferentially inhibited the production of IFN-gamma and blocked IFN-gamma-dependent up-regulation of Fas; however, cytokines such as IL-10, IL-6, or IL-4 were not inhibited. These results suggest that a therapeutic TNF-alpha blockade may promote humoral autoimmunity by selectively inhibiting the induction of a CTL response that would normally suppress autoreactive B cells.