Assessing changes in the surface area of the non-cellulose fraction of lignocellulosics

Cholic acid, which preferentially associates with the non- cellulose component of lignocellulosics, is used as a probe to monitor changes in the relative surface area of this fraction of lignocellulosic biomass-to-ethanol substrates. The method is experimentally demonstrated in a traditional enzyme-catalyzed lignocellulosic saccharification experiment.     

Analysis of hydrostatic pressure-induced changes in umbrella cell surface area

All cells experience and respond to external mechanical stimuli including shear stress, compression, and hydrostatic pressure. Cellular responses can include changes in exocytic and endocytic traffic. An excellent system to study how extracellular forces govern membrane trafficking events is the bladder umbrella cell, which lines the inner surface of the mammalian urinary bladder. It is hypothesized that umbrella cells modulate their apical plasma membrane surface area in response to hydrostatic pressure. Understanding the mechanics of this process is hampered by the lack of a suitable model system. We describe a pressure chamber that allows one to increase hydrostatic pressure in a physiological manner while using capacitance to monitor real-time changes in the apical surface area of the umbrella cell. It is demonstrated that application of hydrostatic pressure results in an increase in umbrella cell apical surface area and a change in the morphology of umbrella cells from roughly cuboidal to squamous. This process is dependent on increases in cytoplasmic Ca(2+). This system will be useful in further dissecting the mechanotransduction pathways involved in cell shape change and regulation of exocytic and endocytic traffic in umbrella cells.     

Determination of the radius, volume and surface area of plasma lipoproteins using fluorescent probes

The effect of radiationless energy transfer between the fluorescent probes was used to determine the radius, volume and surface area of the blood plasma lipoprotein. Anthracene and p-terphenyl, distributed over the whole volume of lipids of a lipoprotein particle, and HSPH-14 localized on its surface served as energy donor and acceptor probes. Human blood lipoproteins of very low (LVLD), low (LLD2), and high (LHD2 and LHD3) density were studied. All the fluorescence-measured lipoprotein volumes were rather close to the data resulted from the weight analysis. The surface areas were equal to 230, 210, 400 and 330 m2 per 1 g of lipoprotein and the radii--11, 11, 7.5, 7.2 nm, respectively. All the measurements were made in solutions, without any denaturating effects on lipoprotein, its concentration being 1 mg/ml and lower.     

Determination of the surface area and viscosity of membranes in T- and B-lymphocytes by means of fluorescent probes

The surface area of lymphocyte membranes was measured by registering F?rster's energy transfer on fluorescent probes. Pyrene served as donor, 4-(n-hydroxystyryl)-N-tetradecylpiridinium (HSP) was the acceptor. The surface area B-lymphocyte membranes was shown to be 1,2 times larger than that of T-lymphocytes. The mean value of lymphocyte membranes viscosity was measured using the excimerization effect of pyrene. This value was the same in all the cells investigated Fluorescence of the probe 3-methoxybenzanthrone (MBA) was 2-2.5 times higher in B-lymphocytes and was not proportional to the surface area of T- and B-cells membranes. MBA fluorescence may imply some differences in physical structures of these cells which are not connected with the viscosity of their membrane lipid phase.     

Theory relating in vitro and in vivo microdialysis with one or two probes

In this paper, we further develop the general theory of microdialysis by extending the linear model of Bungay et al. to provide a theoretical basis for in vitro and in vivo microdialysis. Specifically, we considered the effect of active clearance processes on in vivo microdialysis, and thereby elaborated the theory of Benveniste et al. to endogenous compounds. We examined the use of steady state tissue diffusion resistance with negligible clearance processes to interpret microdialysis data. The influence of the tissue properties on the in vitro and in vivo recoveries in dual-probe microdialysis was analyzed and we simulated the effect of the operating parameters on dual probe microdialysis performance. We estimated that the minimum clearance rate constant detectable by microdialysis in a quasi-steady state is about 5.5 x 10(-5) s(-1). This minimum rate constant establishes a criterion, below which inhibition of the active clearance processes does not show detectable influences on the microdialysis extraction efficiency.     

Validation of urea as an endogenous reference compound for the in vivo calibration of microdialysis probes

Microdialysis is a widely used experimental technique, which offers the opportunity to measure drug and metabolite concentrations in the interstitial space fluid in animals and humans. However, microdialysis probes need to be calibrated in vivo to obtain a recovery factor, which describes the relative drug transfer across the membrane. Recently, novel time-saving calibration techniques, based on the use of reference compounds, have been developed. In particular, the use of endogenous urea levels has been advocated. In the present study we set out to validate the use of the urea reference technique for microdialysis probe calibration in healthy volunteers, employing glucose and paracetamol as model analytes. Urea calibration was compared with the results of two standard calibration techniques, i.e. the no net flux technique and the retrodialysis technique. For glucose, recovery values, calculated by the urea reference technique differed significantly from those values, which were assessed by the no net flux technique (p < 0.05), whereas for paracetamol recovery values did not differ significantly, albeit a high intramethod variability was observed (CV=66%). As a conclusion, we could not confirm the hypothesis that recovery values calculated by the urea reference technique provide equivalent results compared with standard calibration techniques.     

Preliminary evaluation of several disinfection/sterilization techniques for use with microdialysis probes

This study evaluated the suitability of some disinfection and sterilization methods for use with microdialysis probes. Disinfection or sterilization should minimize the tissue inflammatory reaction and improve the long-term health of rats on study and ensure the quality of data obtained by microdialysis sampling. Furthermore, the treatment should not negatively impact probe integrity or sampling performance. The techniques chosen for evaluation included two disinfection methods (70% ethanol and a commercial contact lens solution) and two sterilization methods (hydrogen peroxide plasma, and e-beam radiation). Linear microdialysis probes treated by these processes were compared to untreated probes removed from the manufacturer's packaging as if sterile (the control group). The probes were aseptically implanted in the livers of rats and monitored for 72 hours. The parameters chosen to evaluate probe performance were relative sample mass recovery and the relative in vivo extraction efficiency of the probe for caffeine. Post mortem bacterial counts and histopathology examination of liver tissue were also conducted. The probes remained intact and functional for the entire study period. The methods tested did not acutely alter the probes although hydrogen peroxide plasma and contact lens solution groups showed reduced extraction efficiencies. Minimal tissue damage was observed surrounding the probes and acute inflammatory reaction was mild to moderate. Low numbers of bacterial colonies from the implantation sites indicates that the health of animals in this study was not impaired. This was also true for the control group (untreated probe).     

Relative solvent accessible surface area predicts protein conformational changes upon binding

Protein interactions are often accompanied by significant changes in conformation. We have analyzed the relationships between protein structures and the conformational changes they undergo upon binding. Based upon this, we introduce a simple measure, the relative solvent accessible surface area, which can be used to predict the magnitude of binding-induced conformational changes from the structures of either monomeric proteins or bound subunits. Applying this to a large set of protein complexes suggests that large conformational changes upon binding are common. In addition, we observe considerable enrichment of intrinsically disordered sequences in proteins predicted to undergo large conformational changes. Finally, we demonstrate that the relative solvent accessible surface area of monomeric proteins can be used as a simple proxy for protein flexibility. This reveals a powerful connection between the flexibility of unbound proteins and their binding-induced conformational changes, consistent with the conformational selection model of molecular recognition.     

Rotational dynamics of surface probes in lipid vesicles

Translational and rotational diffusion of fluorescent molecules on the surface of small biological systems such as vesicles, proteins and micelles depolarize the fluorescence. A recent study has treated the case of the translational dynamics of surface probes (M.M.G. Krishna, R. Das, N. Periasamy and R. Nityananda, J. Chem. Phys., 112 (2000) 8502-8514) using Monte Carlo and theoretical methods. Here we extend the application of the methodologies to apply the case of rotational dynamics of surface probes. The corresponding fluorescence anisotropy decays were obtained using the Monte Carlo simulation methods for the two cases: surface probes undergoing rotational dynamics on a plane and on a sphere. The results were consistent with the theoretical equations which show that Monte Carlo methods can be used to simulate the surface diffusion problems. The anisotropy decay for the rotational diffusion of a molecule on a planar surface is single exponential and the residual anisotropy is zero. However, residual anisotropy is finite for the case of rotational diffusion on a sphere because of the spatial averaging of the anisotropy function. The rotational correlation time in both the cases is (4Drot)(-1) with Drot being the rotational diffusion coefficient. Rotational dynamics of a surface bound dye in a single giant liposome and in sonicated vesicles were studied and the results were explained according to the theoretical equations. A fast component of fluorescence depolarization was also observed for sonicated vesicles which was interpreted as wobbling-in-cylinder dynamics of the surface-bound dye.     

Application of light-cured dental adhesive resin for mounting electrodes or microdialysis probes in chronic experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.     

Morphometric changes in the duodenal microvillous surface area of the non-pregnant, pregnant and lactating female rat

In the present study an attempt has been made to describe the morphometrical changes occurring in the microvilli of the duodenum of rats of different ages and periods of reproduction. The data showed that the microvillous surface area in the duodenum was greatest in senescent rats and lowest in adult and old animals. The values for lactating rats were similar to that for the senescent group. There was a direct correlation between the microvillous surface area with the height and density. By contrast, however, there was a negative correlation between the width and density of the duodenal microvilli.     

Decomposing the process of species accumulation into area dependent and time dependent parts

In 1960, Preston predicted that the process of species accumulation in time (species–time relationship, STR) should be similar to the species–area relationship (SAR) and follow a power function with a slope of about 0.26. Here these two conjectures are tested using data of the spatiotemporal species accumulation in a local community of beech forest Hymenoptera. A power function species–area–time model of the form S = S₀ A^z t gave better fits to observed species numbers than a simple power function SAR model, and was able to predict similar species turnover rates (about 9% per year) to those inferred by other methods. The STR was well fitted by a power function, although due the limited time span (8 years) a logarithmic STR pattern cannot be ruled out. STR slopes ranged between 0.01 and 0.23 and were lower than predicted by Preston. Temporal species turnover appeared to be negatively correlated to species densities and positively correlated to species body weights. Ecological guild and taxon membership did not significantly influence temporal species turnover.     

The use of microdialysis to monitor rapid changes in glucose concentration.

As a result of the Diabetes Control and Complications Trial, there is increased emphasis on the importance of blood glucose concentration self-monitoring for people with diabetes. The current methods for this are not ideal, and there are many other possible techniques currently under investigation. One of these techniques is microdialysis, which can be used to analyse subcutaneous interstitial glucose concentrations. A system with high recovery has recently been used to monitor glucose concentrations with sampling over one- or two-hour periods. We have investigated whether this system can be used to monitor rapid changes in blood glucose concentration in healthy volunteers with collection intervals of only ten minutes. The results show that microdialysis can be used to monitor rapidly changing blood glucose concentration, but in some subjects, dialysate glucose lagged behind the whole blood and plasma glucose concentrations to a degree that would be clinically significant. It would therefore be necessary to assess the system, comparing dialysate with plasma glucose concentrations in each individual, prior to use in a clinical setting.     

Energy dependent changes in the cytochromes of the mitochondrial respiratory chain

In intact mitochondria a stoichiometric coupling exists between cytochrome a₃ and the hydrolysis of adenosine triphosphate (ATP). In each case the modification of one cytochrome a₃ (measured as a spectral change) is coupled to the hydrolysis of one ATP molecule. When both cytochromes a₃ and a are reduced the measured equilibrium constant is 0.06 m^?1 but this constant is 10³ M^?1 when both cytochromes are oxidized. When the sixth ligand for cytochrome a₃ is an externally added ligand (HCN, H₂S, CO, NO) the equilibrium constant is different for each ligand, suggesting that the ATP induced modification is of the fifth ligand but that it is energetically dependent on the chemical nature of the sixth ligand. The measured half-reduction potentials for cytochromes a₃ and b_T are dependent on the concentrations of added ATP, adenosine diphosphate (ADP), and orthophosphate. The relationship is consistent with a ligand exchange mechanism in which the ligand on the cytochrome is dependent on the phosphate potential ($\text{ATP}\text{ADP}$ × P_i). The equilibrium constants obtained by the ligand exchange treatment of the E_m values for cytochrome a₃ are consistent with those obtained by direct measurement of the equilibrium constants for the spectrally measured changes.     

Fusion and fission of plasma-membrane material accommodates for osmotically induced changes in the surface area of guard-cell protoplasts

Stomatal movement requires large and repetitive changes in cell volume and consequently changes in surface area. The patch-clamp technique was used to monitor changes in plasma-membrane surface area of individual guard-cell protoplasts (GCPs) by measuring membrane capacitance (C_m), a parameter proportional to the surface area. The membrane capacitance increased under hypoosmotic conditions and decreased after hypertonic treatment. As the specific capacitance remained constant, this demonstrates that osmotically induced changes in surface area are associated with incorporation and removal of membrane material. Osmotically induced fusion and fission of plasma-membrane material was not affected by removal of extracellular Ca²⁺. Dialysing protoplasts with very low (<2 nM) or high (1 μM) Ca²⁺ had no effect on changes in C_m under hypo- and hyperosmotic conditions. However, the rate of change in surface area was dependent on the size of the difference in osmotic potential applied. The larger the osmotic difference and thus changes in membrane tension caused by water influx or efflux, the faster the change in C_m. The results therefore demonstrate that osmotically induced fusion and fission of plasma-membrane material in GCPs are Ca²⁺-independent and modulated by membrane tension. Received: 10 February 1998 / Accepted: 21 April 1998     

Centrosome attachment to the C. elegans male pronucleus is dependent on the surface area of the nuclear envelope

A close association must be maintained between the male pronucleus and the centrosomes during pronuclear migration. In C. elegans, simultaneous depletion of inner nuclear membrane LEM proteins EMR-1 and LEM-2, depletion of the nuclear lamina proteins LMN-1 or BAF-1, or the depletion of nuclear import components leads to embryonic lethality with small pronuclei. Here, a novel centrosome detachment phenotype in C. elegans zygotes is described. Zygotes with defects in the nuclear envelope had small pronuclei with a single centrosome detached from the male pronucleus. ZYG-12, SUN-1, and LIS-1, which function at the nuclear envelope with dynein to attach centrosomes, were observed at normal concentrations on the nuclear envelope of pronuclei with detached centrosomes. Analysis of time-lapse images showed that as mutant pronuclei grew in surface area, they captured detached centrosomes. Larger tetraploid or smaller histone::mCherry pronuclei suppressed or enhanced the centrosome detachment phenotype respectively. In embryos fertilized with anucleated sperm, only one centrosome was captured by small female pronuclei, suggesting the mechanism of capture is dependent on the surface area of the outer nuclear membrane available to interact with aster microtubules. We propose that the limiting factor for centrosome attachment to the surface of abnormally small pronuclei is dynein.     

Hofmeister effects in protein unfolding kinetics: estimation of changes in surface area upon formation of the transition state

We studied the effect of three electrolytes (LiCl, Na(2)SO(4), GuHCl) on the unfolding reaction of chymopapain, a two-domain protein belonging in the papain family of cysteine proteinases. Due to methodological reasons, these studies were carried out at pH 1.5 where the protein unfolds following biphasic kinetics. We have observed the presence of two different effects of electrolyte concentration on the unfolding reactions. At low ionic strength, the ionic atmosphere brought about an increase in reaction rates, regardless of the type of ions being present; this effect is attributed to a general "electrostatic screening" of charge-charge interactions in the macromolecule. At high ionic strength, each electrolyte exerted a distinctively different effect: both rate constants were largely increased by GuHCl (a well-known protein denaturant), but only slightly by LiCl; in contrast, Na(2)SO(4) (a good precipitant) decreased the value of both unfolding rates. These ion-specific (Hofmeister) effects were further used to estimate changes in accessible surface area (DeltaASA) upon formation of the transition states (TS) for unfolding. Results obtained with LiCl and Na(2)SO(4), which we analyzed by means of a parameterization derived from published solubility data of amino acid derivatives, are consistent with DeltaASA increments (for each phase) of about 8.0% of the total theoretical DeltaASA for complete unfolding of the chymopapain molecule. Results in the presence of GuHCl, which were analyzed by using a previous parameterization of protein unfolding data, gave larger DeltaASAs of activation, equivalent to 13 and 16% of the total unfolding DeltaASA.     

pH probes respond to redox changes in cytochrome o

N-Phenylnaphthylamine (NPN) has been used previously to probe the fluidity or microviscosity of membrane lipids. We have shown (Sedgwick, E. G., and Bragg, P.D. (1988) FEBS Lett. 229, 127-130) that the fluorescence intensity of this probe abruptly increases upon depletion of the oxygen content of a medium by respiring cytochrome o of Escherichia coli that has been incorporated into soybean phospholipid vesicles. We now show that the pH probes pyranine and quinacrine behave similarly to NPN. The fluorescence change is not due to changes in the pH gradient across the membrane or to a change in the distribution of probe between the vesicles and the external medium. It is insensitive to uncouplers. The fluorescence change with pyranine and quinacrine occurs also with soluble cytochrome o in the absence of added phospholipid. The NPN response requires added phospholipid. Alteration of the redox state of cytochrome o with cyanide suggests that these probes respond to a change in the redox state of the cytochrome, either by alterations in binding of the probe to the cytochrome or by a change in the environment of the probe bound to the cytochrome. This behavior should be considered when pyranine or quinacrine are used to measure changes in the internal pH of membrane vesicles containing redox proteins.     

Intraannual and interannual changes in the surface area of a closed lake complex in southwestern Siberia using NOAA images

The Lake Chany complex, located in southwestern Siberia, consists of large shallow lakes with an average depth of about 2.2m. The area of the lake fluctuates with the water level, which is closely related to the amount of inflow and evaporation, as the lake complex is endorheic. Using National Oceanic and Atmospheric Administration (NOAA) satellite images of the ice-free periods from 1999 to 2004, we evaluated the seasonal changes in the lake area and surrounding vegetation of the Lake Chany complex. The maximum lake area was observed in early May and decreased until late July. The lake area in August was about 60% of the maximum. Subsequently, the area tended to increase until early October. Compared to ground-truth data, the areas undergoing seasonal fluctuations in the NOAA images corresponded to vast stands of vegetation (Phragmites australis) that were several kilometers wide. These areas seem to be influenced by the inflow of snow-melt water and reed growth. Although interannual differences in the seasonal change in lake area were not great for the whole lake complex, the isolated Yudinskii Pool showed large interannual differences during summer and autumn, suggesting that monitoring this area using NOAA images will be useful for estimating the interannual fluctuations in the southwestern Siberian environment.