Three-dimensional dynamic structure of the liquid-ordered domain in lipid membranes as examined by pulse-EPR oxygen probing

Membranes made of dimyristoylphosphatidylcholine and cholesterol, one of the simplest paradigms for the study of liquid ordered-disordered phase separation, were investigated using a pulse-EPR spin-labeling method in which bimolecular collision of molecular oxygen with the nitroxide spin label is measured. This method allowed discrimination of liquid-ordered, liquid-disordered, and solid-ordered domains because the collision rates (OTP) differ in these domains. Furthermore, the oxygen transport parameter (OTP) profile across the bilayer provides unique information about the three-dimensional dynamic organization of the membrane domains. First, the OTP in the bilayer center in the liquid-ordered domain was comparable to that in the liquid-disordered domain without cholesterol, but the OTP near the membrane surface (up to carbon 9) was substantially smaller in the ordered domain, i.e., the cholesterol-based liquid-ordered domain is ordered only near the membrane surface, still retaining high levels of disorder in the bilayer center. This property may facilitate lateral mobility in ordered domains. Second, in the liquid-disordered domain, the domains with ~5 mol % cholesterol exhibited higher OTP than those without cholesterol, everywhere across the membrane. Third, the transmembrane OTP profile in the liquid-ordered domain that contained 50 mol % cholesterol dramatically differed from that which contained 27 mol % cholesterol.     

Characterization of cholesterol-sphingomyelin domains and their dynamics in bilayer membranes

Lipids segregate with each other into small domains in biological membranes, which can facilitate the associations of particular proteins. The segregation of cholesterol and sphingomyelin (SPM) into domains known as rafts is thought to be especially important. The formation of rafts was studied by using planar bilayer membranes that contained rhodamine-phosphatidylethanolamine (rho-DOPE) as a fluorescent probe, and wide-field fluorescence microscopy was used to detect phase separation of the probe. A fluorescently labeled GM(1), known to preferentially partition into rafts, verified that rho-DOPE faithfully reported the rafts. SPM-cholesterol domains did not form at high temperatures but spontaneously formed when temperature was lowered to below the melting temperature of the SPM. Saturated acyl chains on SPMs therefore promote the formation of rafts. The domains were circular (resolution > or = 0.5 microm), quickly reassumed their circular shape after they were deformed, and merged with each other to create larger domains, all phenomena consistent with liquid-ordered (l(o)) rather than solid-ordered (s(o)) domains. A saturated phosphatidylcholine (PC), disteoryl-PC, could substitute for SPM to complex with cholesterol into a l(o)-domain. But in the presence of cholesterol, a saturated phosphatidylethanolamine or phosphatidylserine yielded s(o)-domains of irregular shape. Lipids with saturated acyl chains can therefore pack well among each other and with cholesterol to form l(o)-domains, but domain formation is dependent on the polar headgroup of the lipid. An individual raft always extended through both monolayers. Degrading cholesterol in one monolayer with cholesterol oxidase first caused the boundary of the raft to become irregular; then the raft gradually disappeared. The fluid nature of rafts, demonstrated in this study, may be important for permitting dynamic interactions between proteins localized within rafts.     

Aeroecology: probing and modeling the aerosphere

Aeroecology is a discipline that embraces and integrates thedomains of atmospheric science, ecology, earth science, geography,computer science, computational biology, and engineering. Theunifying concept that underlies this emerging discipline isits focus on the planetary boundary layer, or aerosphere, andthe myriad of organisms that, in large part, depend upon thisenvironment for their existence. The aerosphere influences bothdaily and seasonal movements of organisms, and its effects haveboth short- and long-term consequences for species that usethis environment. The biotic interactions and physical conditionsin the aerosphere represent important selection pressures thatinfluence traits such as size and shape of organisms, whichin turn facilitate both passive and active displacements. Theaerosphere also influences the evolution of behavioral, sensory,metabolic, and respiratory functions of organisms in a myriadof ways. In contrast to organisms that depend strictly on terrestrialor aquatic existence, those that routinely use the aerosphereare almost immediately influenced by changing atmospheric conditions(e.g., winds, air density, precipitation, air temperature),sunlight, polarized light, moon light, and geomagnetic and gravitationalforces. The aerosphere has direct and indirect effects on organisms,which often are more strongly influenced than those that spendsignificant amounts of time on land or in water. Future advancesin aeroecology will be made when research conducted by biologistsis more fully integrated across temporal and spatial scalesin concert with advances made by atmospheric scientists andmathematical modelers. Ultimately, understanding how organismssuch as arthropods, birds, and bats aloft are influenced bya dynamic aerosphere will be of importance for assessing, andmaintaining ecosystem health, human health, and biodiversity.     

Simulation of toluene decomposition in a pulse-periodic discharge operating in a mixture of molecular nitrogen and oxygen

The kinetic model of toluene decomposition in nonequilibrium low-temperature plasma generated by a pulse-periodic discharge operating in a mixture of nitrogen and oxygen is developed. The results of numerical simulation of plasma-chemical conversion of toluene are presented; the main processes responsible for C₆H₅CH₃ decomposition are identified; the contribution of each process to total removal of toluene is determined; and the intermediate and final products of C₆H₅CH₃ decomposition are identified. It was shown that toluene in pure nitrogen is mostly decomposed in its reactions with metastable N₂(A₃?? _u ⁺ ) and N₂(a??¹?? _u ^? ) molecules. In the presence of oxygen, in the N₂ : O₂ gas mixture, the largest contribution to C₆H₅CH₃ removal is made by the hydroxyl radical OH which is generated in this mixture exclusively due to plasma-chemical reactions between toluene and oxygen decomposition products. Numerical simulation showed the existence of an optimum oxygen concentration in the mixture, at which toluene removal is maximum at a fixed energy deposition.     

Hopanoids in marine cyanobacteria: probing their phylogenetic distribution and biological role

Cyanobacteria are key players in the global carbon and nitrogen cycles and are thought to have been responsible for the initial rise of atmospheric oxygen during the Neoarchean. There is evidence that a class of membrane lipids known as hopanoids serve as biomarkers for bacteria, including many cyanobacteria, in the environment and in the geologic record. However, the taxonomic distributions and physiological roles of hopanoids in marine cyanobacteria remain unclear. We examined the distribution of bacteriohopanepolyols (BHPs) in a collection of marine cyanobacterial enrichment and pure cultures and investigated the relationship between the cellular abundance of BHPs and nitrogen limitation in Crocosphaera watsonii, a globally significant nitrogen‐fixing cyanobacterium. In pure culture, BHPs were only detected in species capable of nitrogen fixation, implicating hopanoids as potential markers for diazotrophy in the oceans. The enrichment cultures we examined exhibited a higher degree of BHP diversity, demonstrating that there are presently unaccounted for marine bacteria, possibly cyanobacteria, associated with the production of a range of BHP structures. Crocosphaera watsonii exhibited high membrane hopanoid content consistent with the idea that hopanoids have an important effect on the bulk physical properties of the membrane. However, the abundance of BHPs in C. watsonii did not vary considerably when grown under nitrogen‐limiting and nitrogen‐replete conditions, suggesting that the role of hopanoids in this organism is not directly related to the physiology of nitrogen fixation. Alternatively, we propose that high hopanoid content in C. watsonii may serve to reduce membrane permeability to antimicrobial toxins in the environment.     

DNA稳定同位素探针技术在宏基因组学中的应用及前景

DNA稳定同位素探针 (DNA-SIP) 是一种新兴的技术,通过将同位素稳定结合到特定的底物来确定环境中微生物的作用。DNA-SIP与宏基因组学结合可以让某些微生物的特性与其特殊新陈代谢联系在一起,不仅可以从宏基因组库里检测到低含量的微生物,而且加速了对新的酶类和其他生物活性物质的发现。以下总结了SIP-宏基因组学技术的原理、应用及研究进展,并讨论了其在环境微生物学和生物技术的应用前景。     

Circuit-breakers: optical technologies for probing neural signals and systems

Neuropsychiatric disorders, which arise from a combination of genetic, epigenetic and environmental influences, epitomize the challenges faced in understanding the mammalian brain. Elucidation and treatment of these diseases will benefit from understanding how specific brain cell types are interconnected and signal in neural circuits. Newly developed neuroengineering tools based on two microbial opsins, channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), enable the investigation of neural circuit function with cell-type-specific, temporally accurate and reversible neuromodulation. These tools could lead to the development of precise neuromodulation technologies for animal models of disease and clinical neuropsychiatry.     

Oligopeptidase B: Cloning and probing stability under nonequilibrium conditions

Oligopeptidase B is a member of a new serine peptidase family, unrelated to the trypsin and subtilisin families. It is a potential processing enzyme of prokaryotes, being very specific for the basic amino acid pairs of polypeptides. An understanding of the kinetics of the enzyme requires the examination of its conformational stability under a variety of conditions. To this end, the enzyme was cloned from Escherichia coli HB101 by the PCR method, expressed with high yield in E. coli XL1-Blue, and purified essentially in two chromatographic steps. The denatured enzyme failed to refold, which precluded the calculation of free energy of stability, ΔG₀. Therefore, the unfolding rates were measured to probe the stability against urea, pH, and heat. Denaturation processes were monitored by intrinsic fluorescence, circular dichroism, and activity measurements. A static method, intrinsic fluorescence vs. pH, was indicative of significant changes in the tertiary structure of the enzyme pH < 6 and pH > 8.5. The more sensitive dynamic methods, unfolding rates in urea and inactivation rates at high temperature, revealed increased flexibility in the protein structure between pH 6 and pH 7, where the static method did not show significant changes. Inactivation of the enzyme in the acidic pH range correlated with the results obtained with the static rather than with the dynamic method. Acid denaturation at pH 3 was markedly retarded by 1 M NaCl. Against heat inactivation the enzyme was also considerably protected in the presence of salt, and the higher enthalpy and entropy of activation suggested the importance of hydration in the stabilization. The kinetics of unfolding followed single-exponential decay under strongly denaturing conditions (high urea concentration or high temperature), but deviated from the apparently two-state mechanism at low urea concentrations and at slightly acidic pH. The results indicate that under harsher denaturing conditions there is a single rate-limiting step in unfolding, whereas under milder conditions partly unfolded intermediates are populated. Proteins 30:424–434, 1998. © 1998 Wiley-Liss, Inc.     

Single-cell unroofing: probing topology and nanomechanics of native membranes

Cell membranes separate the cell interior from the external environment. They are constituted by a variety of lipids; their composition determines the dynamics of membrane proteins and affects the ability of the cells to adapt. Even though the study of model membranes allows to understand the interactions among lipids and the overall mechanics, little is known about these properties in native membranes. To combine topology and nanomechanics analysis of native membranes, I designed a method to investigate the plasma membranes isolated from a variety of single cells. Five cell types were chosen and tested, revealing 20% variation in membrane thickness. I probed the resistance of the isolated membranes to indent, finding their line tension and spreading pressure. These results show that membranes isolated from neurons are stiffer and less diffusive than brain cancer cell membranes. This method gives direct quantitative insights on the mechanics of native cell membranes.