Cell-to-cell transfer of glial proteins to the squid giant axon: The glia- neuron protein transfer hypothesis

The hypothesis that glial cells synthesize proteins which are transferred to adjacent neurons was evaluated in the giant fiber of the squid (Loligo pealei). When giant fibers are separated from their neuron cell bodies and incubated in the presence of radioactive amino acids, labeled proteins appear in the glial cells and axoplasm. Labeled axonal proteins were detected by three methods: extrusion of the axoplasm from the giant fiber, autoradiography, and perfusion of the giant fiber. This protein synthesis is completely inhibited by puromycin but is not affected by chloramphenicol. The following evidence indicates that the labeled axonal proteins are not synthesized within the axon itself. (a) The axon does not contain a significant amount of ribosomes or ribosomal RNA. (b) Isolated axoplasm did not incorporate [(3)H]leucine into proteins. (c) Injection of Rnase into the giant axon did not reduce the appearance of newly synthesized proteins in the axoplasm of the giant fiber. These findings, coupled with other evidence, have led us to conclude that the adaxonal glial cells synthesize a class of proteins which are transferred to the giant axon. Analysis of the kinetics of this phenomenon indicates that some proteins are transferred to the axon within minutes of their synthesis in the glial cells. One or more of the steps in the transfer process appear to involve Ca++, since replacement of extracellular Ca++ by either Mg++ or Co++ significantly reduces the appearance of labeled proteins in the axon. A substantial fraction of newly synthesized glial proteins, possibly as much as 40 percent, are transferred to the giant axon. These proteins are heterogeneous and range in size from 12,000 to greater than 200,000 daltons. Comparisons of the amount of amino acid incorporation in glia cells and neuron cell bodies raise the possibility that the adaxonal glial cells may provide an important source of axonal proteins which is supplemental to that provided by axonal transport from the cell body. These findings are discussed with reference to a possible trophic effect of glia on neurons and metabolic cooperation between adaxonal glia and the axon.     

Inhibition of Mitochondrial ATP Generation by Nitric Oxide Switches Apoptosis to Necrosis

Under pathological conditions, the mode of cell death, apoptosis or necrosis, is relevant for the subsequent fate of the tissue. Cell demise may be shaped by endogenous mediators such as nitric oxide (NO) which interfere with subroutines of the death program. Here we show that apoptosis of Jurkat cells elicited by either staurosporine (STS) or anti-CD95 antibodies in glucose-free medium is converted to necrosis by NO donors. In the presence of NO, release of mitochondrial cytochrome c was delayed and activation of execution caspases was prevented. Stimulated cells died nonetheless. The switch in the mode of cell death was due to NO-dependent failure of mitochondrial energy production. Restoration of intracellular ATP by glucose supplementation recovered the cells' ability to activate caspases and undergo apoptosis. In this system, the apoptosis/necrosis conversion promoted by NO was not mediated by cyclic guanosine monophosphate-dependent mechanisms, poly-(ADP-ribose)-polymerase (PARP) activation, or inhibition of caspases due to S-nitrosylation and glutathione depletion. In contrast, depleting intracellular ATP with rotenone, an inhibitor of mitochondrial complex I mimicked the effect of NO. The findings presented here suggest that NO can decide the shape of cell death by lowering intracellular ATP below the level required to allow the coordinated execution of apoptosis.     

A modular treatment of molecular traffic through the active site of cholinesterase

We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.     

哈尔滨西郊赤狐冬季巢区的初步研究

本文利用雪地跟踪方法对哈尔滨西郊5只赤狐在1985-1986年冬季的巢区做了观察。结果表明,5只狐对巢区内各部分使用的强度是不等的,对巢区中部的某些地块使用强度要高于对外围的使用,并具有明显的方向性。5个巢区的平均活动半径为320±68米至557±82米,面积为1.44-4.O9平方公里,线性指数为1.079至2。5只狐相邻距离约1000米。     

Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells

A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol rather than Glc3Man9GlcNAc2-P-P-dolichol as the oligosaccharide-lipid intermediate in N-glycosylation of proteins. MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol. The mannose-labeled oligosaccharide released from oligosaccharide-lipid of MI8-5 cells was analyzed by HPLC and alpha-mannosidase treatment, and the data were consistent with a structure of Man9GlcNAc2. In addition, MI8-5 cells did not incorporate radioactivity into oligosaccharide- lipid during an incubation with tritiated galactose, again consistent with MI8-5 cells synthesizing an unglucosylated oligosaccharide-lipid. MI8-5 cells had parental levels of glucosylphosphoryldolichol synthase activity. However, in two different assays, MI8-5 cells lacked dolichol- P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase activity. MI8-5 cells were found to synthesize glucosylated oligosaccharide after they were transfected with Saccharomyces cerevisiae ALG 6, the gene for dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase. MI8-5 cells were found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.      

Dehalogenation of dichloroethene in a contaminated soil: fatty acids and alcohols as electron donors and an apparent requirement for tetrachloroethene

Environmental soil contamination at an industrial site in Marion, Ohio (USA) with tetrachloroethene (perchloroethene, PCE) resulted in residual cis-1, 2-dichloroethene (DCE) contamination that had not declined after more than 15 years. Microcosm slurries containing 2.6% soil from this site were supplemented with different electron donors, i.e., individual fatty acids or alcohols. None of the microcosms supported complete DCE dechlorination, unless PCE was added to the microcosm at initiation. The addition of fresh PCE resulted in the dehalogenation of PCE to DCE in the microcosms supplemented with fatty acids having an even number of carbon atoms (acetate, butyrate, and caproate), but not in those with an odd number of carbon atoms (formate, propionate, and valerate), where negligible or no activity was detected. No significant further DCE degradation was observed in any of the microcosms supplied with fatty acids as electron donors. Microcosms supplemented with freshly added PCE bioconverted PCE to DCE and completely dehalogenated both the ex-novo and soil-supplied DCE within 60 days, but only if alcohols having an even number of carbon atoms (ethanol or butanol) were also added as electron donors. Odd-numbered alcohols either did not produce dehalogenation (as with methanol) or only dehalogenated PCE to DCE (as with propanol).     

Soya lecithin effects on the aerobic biodegradation of polychlorinated biphenyls in an artificially contaminated soil

The effects of the phytogenic surfactant soya lecithin (SL) on the aerobic biodegradation of polychlorinated biphenyls (PCBs) spiked into a synthetic soil were studied. Soil was spiked with both biphenyl (4 g/kg) and Fenclor 42 (1,000 mg/kg) and treated in aerobic batch slurry-phase microcosms (17.5% w/v). Microcosms were prepared either with or without the exogenous aerobic PCB-dechlorinating bacterial co-culture ECO3 (inoculum:10(8) CFU/mL). In some inoculated microcosms, SL was added at 15 or 30 g/kg. Indigenous bacteria having the capability of metabolizing biphenyl and 2-chlorobenzoic acid were found to develop in the microcosms during the experiment, and were responsible for the significant PCB biodegradation and dechlorination observed in the uninoculated controls. The addition of ECO3 bacteria resulted in only a slight PCB biodegradation increase. In the presence of SL, a higher availability of biphenyl- and chlorobenzoic acid-degrading bacteria and higher PCB biodegradation and dechlorination yields were observed; the effects increased proportionally with the concentration of the applied SL. A significant decrease of soil ecotoxicity was also revealed in SL-supplemented microcosms. At both concentrations, SL was found to be a good carbon source for both the indigenous and ECO3 bacteria, as well as a product capable of enhancing the PCB bioavailability in the microcosms.     

Aerobic mineralization of chlorobenzoates by a natural polychlorinated biphenyl-degrading mixed bacterial culture

A natural mixed aerobic bacterial culture, designated MIXE1, was found to be capable of degrading several low-chlorinated biphenyls when 4-chlorobiphenyl was used as a co-substrate. MIXE1 was capable of using all the three monochlorobenzoate (CBA) isomers tested as well as 2,5-, 3,4- and 3,5-dichlorobenzoate (dCBA) as the sole carbon and energy source. During MIXE1 growth on these substrates, a nearly stoichiometric amount of chloride was released: 0.5 g/l of each chlorobenzoate was completely mineralized by MIXE1 after 2 or 3 days of culture incubation. Two strains, namely CPE2 and CPE3, were selected from MIXE1: CPE2, referred to the Pseudomonas genus, was found to be capable of totally degrading both 2-CBA and 2,5-dCBA, whereas Alcaligenes strain CPE3 was capable of mineralizing 3-, 4-CBA and 3,4-dCBA. Substrate uptake studies carried out with whole cells of strain CPE2 suggested that 2-CBA was metabolized through catechol, while 2,5-dCBA was degraded via 4-chlorocatechol. 3-CBA, 4-CBA, and 3,4-dCBA appeared to be degraded through 3,4-dihydroxybenzoate by the CPE3 strain. MIXE1, which is capable of degrading several chlorobenzoates, should therefore be able to mineralize a number of low-chlorinated congeners of simple and complex polychlorinated biphenyl mixtures. Correspondence to: F. Fava