Entropic interaction chromatography: separating proteins on the basis of size using end-grafted polymer brushes

Partitioning of a macromolecule into the interfacial volume occupied by a grafted polymer brush decreases the configurational entropy (DeltaSbrush(c)) of the terminally attached linear polymer chains due to a loss of free volume. Self-consistent field theory (SCF) calculations are used to show that DeltaSbrush(c) is a strong function of both the size (MWp) of the partitioning macromolecule and the depth of penetration into the brush volume. We further demonstrate that the strong dependence of DeltaSbrush(c) on MWp provides a novel and powerful platform, which we call entropic interaction chromatography (EIC), for efficiently separating mixtures of proteins on the basis of size. Two EIC columns, differing primarily in polymer grafting density, were prepared by growing a brush of poly(methoxyethyl acrylamide) chains on the surface of a wide-pore (1,000-A pores, 64-microm diameter rigid beads) resin (Toyopearl AF-650M) bearing surface aldehyde groups. Semipreparative 0.1-L columns packed with either EIC resin provide reduced-plate heights of 2 or less for efficient separation of globular protein mixtures over at least three molecular-weight decades. Protein partitioning within these wide-pore EIC columns is shown to be effectively modeled as a thermodynamically controlled process, allowing partition coefficients (K(P)) and elution chromatograms to be accurately predicted using a column model that combines SCF calculation of K(P) values with an equilibrium-dispersion type model of solute transport through the column. This model is used to explore the dependence of column separation efficiency on brush properties, predicting that optimal separation of proteins over a broad MWp range is achieved at low to moderate grafting densities and intermediate chain lengths.     

Functional overexpression and in vitro re-association of OpuA, an osmotically regulated ABC-transport complex from Bacillus subtilis

The osmotically regulated OpuA uptake system from Bacillus subtilis is a member of the SBP-dependent subfamily of ABC-transporters. The functional complex, OpuA(A(2)B(2)C), catalyzes the osmotically controlled import of the compatible solutes glycine betaine and proline betaine. Here, we describe the purification of the isolated TMS, OpuAB. Stimulated ATPase activity of OpuAA by OpuAB demonstrated that OpuAB adopts a functional fold. An interaction between all subunits could be verified in detergent solution with the highest ATPase stimulation determined for the dimeric NBS in the re-associated complex in the presence of all transport components plus substrate.     

Attenuated phosphorylation of heat shock protein 27 correlates with tumor progression in patients with hepatocellular carcinoma

Heat shock protein 27 (HSP27) is expressed at high levels in human hepatocellular carcinoma (HCC). We examined correlations of total HSP27 and serine phosphorylated (Ser-15, Ser-78, and Ser-82) HSP27 levels in HCC tissues with clinical and pathologic characteristics in 48 resected HCC specimens. The levels of total and Ser-phosphorylated HSP27 were evaluated by Western blot analysis. Immunohistochemical analysis of HSP27 expression was also performed on some samples. Phosphorylation of HSP27 was detected in all 48 HCC tissues. Levels of phosphorylated HSP27 were correlated inversely with tumor size, microvascular invasion of HCC, and tumor stage by TNM classification. In contrast, only microvascular invasion showed an inverse correlation with total HSP27 levels. The decrease in phosphorylated HSP27 in progressed HCC was also observed by immunohistochemistry. Levels of phosphorylated HSP27 gradually decreased in parallel with HCC progression. Our findings suggest that phosphorylated HSP27 may have a suppressive role in progression of human HCC.     

Mandibular premolar and second molar root morphological variation in modern humans: What root number can tell us about tooth morphogenesis

This investigation of modern human mandibular premolar root variation tests the hypothesis that population-specific mandibular single-rooted premolar root size can predict a predisposition to root morphological complexity. Mandibular postcanines were examined and quantified from dental radiographs in a globally spread sample of 1,615 modern humans. Multirooted premolars and a fused molar root phenotype were investigated as probes into greater than, and less than, the normative root number. Twelve questions were addressed concerning root structure of mandibular premolars and second molars. A direct correlation was found between single-rooted mandibular premolar size and the predisposition to multirootedness. This correlation infers the following: 1) that postcanine primordia size during root formation predisposes to the development of more, or less, than the normative postcanine root number; and 2) that the epigenetic effect of tooth primordium size per se influences the induction of interradicular processes, which divides the root during its development. This simple developmental model helps explain the following observations: 1) population-specific variation in postcanine root number; 2) sexual dimorphism for multirooted mandibular premolar prevalence; 3) why microdont teeth are single-rooted; 4) the hierarchy of developmental canalization of interradicular processes; 5) megadont-hominin to late-hominin mandibular premolar root number transition; and 6) the fluctuation of mandibular premolar root number in primate evolutionary history.     

Larval nutrition affects lipid storage and growth, but not protein or carbohydrate storage in newly eclosed adults of the grasshopper Schistocerca americana

Nitrogen availability from dietary protein can have profound effects on the physiology and evolutionary ecology of insect herbivores. While many studies consider the effects of nutrition on consumption and gross body composition of protein and other important nutrients, few consider partitioning to storage for future use. I used chemically defined artificial diets to quantitatively manipulate the amount of dietary carbohydrates and proteins available to growing larvae of the grasshopper Schistocerca americana to determine how larval nutrient availability affects growth and all three classes of stored nutrients (proteins, lipids, and carbohydrates) carried over from larval feeding into adulthood. Larvae on poor diets increased consumption, but could not compensate for diet quality, eclosing small and containing no significant nutrient stores at adulthood. Individuals fed intermediate to high nutrient content diets as larvae were significantly larger and contained a significantly greater proportion of lipid stores at adult eclosion, but not protein or carbohydrate stores than individuals fed low nutrient content diets. This suggests that larvally derived lipid stores may be more important to adult fitness than carbohydrate or protein stores. This result is contrary to previous studies performed on the role of larval nutrition and allocation to protein stores, and this difference is likely due to variation in the relative availability of protein in adult diets across species.     

Relation between insulin resistance and fast-migrating LDL subfraction as characterized by capillary isotachophoresis

The proportion of the electronegative low density lipoprotein [LDL(-)] subfraction, which is atherogenic, is increased in type 2 diabetes but is not reduced by glycemic control. Therefore, we evaluated the ability of a new technique, capillary isotachophoresis (cITP), to quantify charge-based LDL subfractions and examined the relation between insulin resistance and the cITP fast-migrating (f) LDL levels. Seventy-five 10-year-old boys were included. The two cITP LDL subfractions, fLDL and major LDL subfractions, were proportional to the LDL protein content within the range of 0.1-0.8 mg/ml LDL protein. Levels of cITP fLDL were positively correlated with triglyceride (TG) levels and negatively correlated with LDL size. Insulin resistance as assessed by the homeostasis model assessment (HOMA-IR) was positively correlated (P < 0.01) with cITP fLDL levels (r = 0.41). The relation between HOMA-IR and cITP fLDL levels depended on TG levels but was independent of body mass index and LDL size. cITP lipoprotein analysis is an accurate and sensitive method for quantifying charge-based LDL subfractions in human plasma, and insulin resistance is related to cITP fLDL independent of LDL size.     

Relation between cell disruption conditions, cell debris particle size, and inclusion body release

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric peptidoglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity.     

Perspective: the size-complexity rule

It is widely accepted that bigger entities have a greater division of labor than smaller ones and this is reflected in the fact that larger multicellular organisms have a corresponding increase in the number of their cell types. This rule is examined in some detail from very small organisms to large animals, and plants, and societies. Compared to other size-related rules, the size-complexity rule is relatively rough and approximate, yet clearly it holds throughout the whole range of living organisms, as well as for societies. The relationship between size and complexity is analyzed by examining the effects of size increase and decrease: size increase requires an increase in complexity, whereas size decrease permits, and sometimes requires, a decrease in complexity. Conversely, an increase or decrease in complexity permits, but does not require changes in size. An especially compelling argument for the close relation between size and complexity can be found in size quorum sensing in very small multicellular organisms.     

Bigger is not always better: conflicting selective pressures on seed size in Quercus ilex

Most theoretical treatments of the evolutionary ecology of offspring size assume a simple and direct effect of investment per offspring on offspring fitness. In this paper I experimentally determine the relationship between seed mass and several main fitness components of the oak Quercus ilex, to estimate phenotypic selection acting on seed mass during the early life cycle and to discover any potential selective conflicts occurring between different stages from dispersal to establishment. I found a positive effect of acorn size on most fitness components related to seedling establishment. Large size increased germination rate and seedling survival, accelerated germination timing, and enhanced seedling growth. Nevertheless, there was also a direct negative effect of acorn size on survival to predation, because large acorns were highly preferred by the main postdispersal seed predators at the study site, wild boars and wood mice. Because of the low probability of escape from predation, the fitness of large acorns estimated on this component was significantly lower than the fitness of smaller acorns. Therefore, seed size affected fitness in two different ways, yielding opposing and conflicting selective forces. These findings suggest that the general assumption that offspring fitness is a fixed positive function of seed size needs to be reconsidered for some systems. The existence of conflicting selection might explain the occurrence of an optimal seed size in some plant species without invoking a seed number-size trade-off.