Effects of halothane on dipalmitoylphosphatidylcholine liposomes: a Raman spectroscopic study

Raman spectroscopy has been used to monitor the concentration of halothane (1-bromo-1-chloro-2,2,2-trifluoroethane) in 20% aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) as well as to follow changes in the acyl chain order within the hydrocarbon interior of the liposomes. Temperature profiles for the gel to liquid-crystalline phase transitions for the liposomes were constructed from changes in peak height intensity ratios in the C-H stretching mode and C-C stretching mode regions. Halothane present at the clinical level produces a change of -0.5 degrees C in the phase transition temperature. A limiting transition temperature of about 21 degrees C and saturation of the gel phase occur when the molar ratio of halothane to DPPC reaches about 1.25. At molar ratios above 2.1, the liquid-crystalline phase is also saturated with halothane. Calculations of the distribution of halothane between the various phases in the system are presented and used to interpret literature data as well as the present experiments. Ideal solution theory accounts rather well for the depression in the transition temperature over most of the mole ratio range, an outcome which implies that halothane is excluded from the hydrocarbon interior but not the head-group region in the gel phase. The role of halothane in the head-group region is discussed.     

Lipid growth requirement and influence of lipid supplement on fatty acid and aldehyde composition of Syntrophococcus sucromutans

Results concerning the ruminal fluid growth requirement of the ruminal acetogen, Syntrophococcus sucromutans, indicate that octadecenoic acid isomers satisfy this essential requirement. Complex lipids, such as triglycerides and phospholipids, can also support growth. The cellular fatty acid and aldehyde composition closely reflects that of the lipid supplement provided to the cells. Up to 98% of the fatty acids and 80% of the fatty aldehydes are identical in chain length and degree of unsaturation to the octadecenoic acid supplement provided in the medium. S. sucromutans shows a tendency to have a greater proportion of the aldehyde form among its 18 carbon chains than it does with the shorter-chain simple lipids, which may be interpreted as a strategy to maintain membrane fluidity. 14C labeling showed that most of the oleic acid taken up from the medium was incorporated into the membrane fraction of the cells.     

The role of hydrogel structure and dynamic loading on chondrocyte gene expression and matrix formation

Crosslinked poly(ethylene glycol) (PEG) hydrogels are attractive scaffolds for cartilage tissue engineering because of their ability to mimic the aqueous environment and mechanical properties of native cartilage. In this study, hydrogel crosslinking density was varied to study the influence of gel structure and the application of dynamic loading (continuous, 1 Hz, 15% amplitude strain) on chondrocyte gene expression over 1 week culture. Gene expression was quantified using real-time RT-PCR for collagen II and aggrecan, the major cartilage extracellular matrix (ECM) components, and collagen I, an indicator of chondrocyte de-differentiation. When chondrocytes were encapsulated in PEG gels with low or high crosslinking, a high collagen II expression compared to collagen I expression (1000 or 100,000:1, respectively) indicated the native chondrocyte phenotype was retained. In the absence of loading, relative gene expression for collagen II and aggrecan was significantly higher (e.g., 2-fold and 4-fold, respectively, day 7) in the low crosslinked gels compared to gels with higher crosslinking. Dynamic loading, however, showed little effect on ECM gene expression in both crosslinked systems. To better understand the cellular environment, ECM production was qualitatively assessed using an in situ immunofluorescent technique and standard histology. A pericellular matrix (PCM) was observed as early as day 3 post-encapsulation and the degree of formation was dependent on gel crosslinking. These results suggest the PCM may protect the cells from sensing the applied loads. This study demonstrates that gel structure has a profound effect on chondrocyte gene expression, while dynamic loading has much less of an effect at early culture times.     

Hydrochemical fluctuations and crustacean community composition in an ephemeral saline lake (Sua Pan,Makgadikgadi Botswana)

Fluctuating hydrochemistry, as a result of extreme hydrological regimes, imposes major physiological constraints on the biota of ephemeral saline lakes. While the inverse relationship between salinity and zooplankton species richness is well-known across salinity gradients, few studies have documented closely the response of zooplankton to seasonal changes in salinity. Weekly sampling during two flood seasons at Sua Pan, an intermittent saline lake in central Botswana demonstrated the importance of spatial and temporal salinity gradients for crustacean community composition, associated with a decline in species richness, from 11 to three species. Conductivity ranged between 320 and 125,800 μS cm⁻¹ during seasonal flooding; changing from dominance by and , Ca²⁺ and Mg²⁺, at the beginning of the floods, to NaCl dominated waters as the lake dried out and salinities increased. pH estimates generally ranged between 8.6 and 10, with maximum values recorded during initial flooding. Crustaceans comprised mainly Branchinella spinosa, Moina belli, Lovenula africana and Limnocythere tudoranceai, all of which occurred across a wide range of salinities, while halotolerant freshwater species (Metadiaptomus transvaalensis, Leptestheria striatochonca and the ostracods Plesiocypridopsis aldabrae, Cypridopsis newtoni and a newly identified Potamocypris species) disappeared above conductivities of 1,500 μS cm⁻¹. A unique crustacean composition in southern Africa was attributed to Sua Pans’ rare chemical composition among southern African saline lakes; flood waters on Sua Pan contained a higher proportion of Na⁺ and , and less K⁺, Mg²⁺ and than over 80% of records from salt pans elsewhere in southern African. The freshwater species of crustaceans in Sua Pan were similar to those found in other southern Africa lakes, and these similarities decreased in lakes with higher pH and proportions of Na, and less SO₄ and Mg in their chemical composition. The predominant saline tolerant species on Sua Pan, however, showed a greater similarity to those in saline lakes in southern and East Africa with higher proportions of and, particularly, Mg²⁺ in their chemical composition. Handling editor: J. M. Melack     

Monte Carlo simulations of supercoiled DNAs confined to a plane.

Recent advances in atomic force microscopy (AFM) have enabled researchers to obtain images of supercoiled DNAs deposited on mica surfaces in buffered aqueous milieux. Confining a supercoiled DNA to a plane greatly restricts its configurational freedom, and could conceivably alter certain structural properties, such as its twist and writhe. A program that was originally written to perform Monte Carlo simulations of supercoiled DNAs in solution was modified to include a surface potential. This potential flattens the DNAs to simulate the effect of deposition on a surface. We have simulated transfers of a 3760-basepair supercoiled DNA from solution to a surface in both 161 and 10 mM ionic strength. In both cases, the geometric and thermodynamic properties of the supercoiled DNAs on the surface differ significantly from the corresponding quantities in solution. At 161 mM ionic strength, the writhe/twist ratio is 1.20-1.33 times larger for DNAs on the surface than for DNAs in solution and significant differences in the radii of gyration are also observed. Simulated surface structures in 161 mM ionic strength closely resemble those observed by AFM. Simulated surface structures in 10 mM ionic strength are similar to a minority of the structures observed by AFM, but differ from the majority of such structures for unknown reasons. In 161 mM ionic strength, the internal energy (excluding the surface potential) decreases substantially as the DNA is confined to the surface. Evidently, supercoiled DNAs in solution are typically deformed farther from the minimum energy configuration than are the corresponding surface-confined DNAs. Nevertheless, the work (Delta A(int)) done on the internal coordinates, which include uniform rotations at constant configuration, during the transfer is positive and 2.6-fold larger than the decrease in internal energy. The corresponding entropy change is negative, and its contribution to Delta A(int) is positive and exceeds the decrease in internal energy by 3.6 fold. The work done on the internal coordinates during the solution-to-surface transfer is directed primarily toward reducing their entropy. Evidently, the number of configurations available to the more deformed solution DNA is vastly greater than for the less deformed surface-confined DNA.     

Exploring functional roles of multibinding protein interfaces

Cellular processes are highly interconnected and many proteins are shared in different pathways. Some of these shared proteins or protein families may interact with diverse partners using the same interface regions; such multibinding proteins are the subject of our study. The main goal of our study is to attempt to decipher the mechanisms of specific molecular recognition of multiple diverse partners by promiscuous protein regions. To address this, we attempt to analyze the physicochemical properties of multibinding interfaces and highlight the major mechanisms of functional switches realized through multibinding. We find that only 5% of protein families in the structure database have multibinding interfaces, and multibinding interfaces do not show any higher sequence conservation compared with the background interface sites. We highlight several important functional mechanisms utilized by multibinding families. (a) Overlap between different functional pathways can be prevented by the switches involving nearby residues of the same interfacial region. (b) Interfaces can be reused in pathways where the substrate should be passed from one protein to another sequentially. (c) The same protein family can develop different specificities toward different binding partners reusing the same interface; and finally, (d) inhibitors can attach to substrate binding sites as substrate mimicry and thereby prevent substrate binding.