Considerations for operational space definition and optimization of a no-salt flowthrough hydrophobic interaction chromatography purification step

No-salt flowthrough hydrophobic interaction chromatography (HIC) has been shown to effectively remove process and product-related impurities from bioprocess streams. In this publication, a panel of six antibodies has been used to demonstrate operating principles for the application of no-salt flowthrough HIC in antibody purification processes. The results indicate that no-salt flowthrough HIC provides robust aggregate clearance across operating conditions including flow rate, and variations in resin ligand density. Additionally, HMW reduction has an optimal pH range relative to the isoelectric point of each molecule and high molecular weight (HMW) reduction can be improved by altering the total protein load and/or HMW concentration to drive binding of high molecular weight species to the resin.     

Dietary patterns,abdominal visceral adipose tissue,and cardiometabolic risk factors in African Americans: The Jackson heart study

Objective:

To examine the relative association of abdominal visceral adipose tissue (VAT) with cardiometabolic risk factors between African and European Americans.

Design and Methods:

We conducted a cross‐sectional study of 2035 African Americans from Jackson Heart Study (JHS) and 3170 European Americans from Framingham Heart Study (FHS) who underwent computed tomography assessment of VAT and subcutaneous adipose tissue (SAT). The FHS participants were weighted to match the age distribution of the JHS participants and the metabolic risk factors were examined by study groups in relation to VAT.

Results:

JHS participants had higher rates of obesity, hypertension, diabetes and metabolic syndrome than FHS participants (all p = 0.001). The associations were weaker in JHS women for VAT with blood pressure, triglycerides, HDL‐C, and total cholesterol (p_interaction = 0.03 to 0.001) than FHS women. In contrast, JHS men had stronger associations for VAT with high triglycerides, low HDL, and metabolic syndrome (all p_interaction = 0.001) compared to FHS men. Similar associations and gender patterns existed for SAT with most metabolic risk factors.

Conclusions:

The relative association between VAT and cardiometabolic risk factors is weaker in JHS women compared to FHS women, whereas stronger association with triglycerides and HDL were observed in JHS men.     

Conformational restriction in a series of GPR119 agonists: Differences in pharmacology between mouse and human

A series of conformationally restricted GPR119 agonists were prepared based around a 3,8-diazabicyclo[3.2.1]octane scaffold. Examples were found to have markedly different pharmacology in mouse and human despite similar levels of binding to the receptor. This highlights the large effects on GPCR phamacology that can result from small structural changes in the ligand, together with inter-species differences between receptors.     

Towards normalization of soybean somatic embryo maturation

Soybean (Glycine max L. Merrill) somatic embryos have been useful for assaying seed-specific traits prior to plant recovery. Such traits could be assessed more accurately if somatic embryos more closely mimicked seed development. Amino acid supplements, carbon source, and abscisic acid and basal salt formulations were tested in an effort to modify existing soybean embryogenesis histodifferentiation/maturation media to further normalize the development of soybean somatic embryos. The resultant liquid medium, referred to as soybean histodifferentiation and maturation medium (SHaM), consists of FNL basal salts, 3% sucrose, 3% sorbitol, filter-sterilized 30 mM glutamine and 1 mM methionine. SHaM-derived somatic embryos are more similar to seed in terms of protein and fatty acid/lipid composition, and conversion ability, than somatic embryos obtained from traditional soybean histodifferentiation and maturation media.     

Cortical bone remodeling rates in a sample of African American and European American descent groups from the American Midwest: comparisons of age and sex in ribs

This study employs regression analysis to explore population and sex differences in the pattern of age-associated bone loss, as reflected by histomorphometric variables that are measures of intracortical and endocortical bone remodeling. A comparison of an African American sample from the Washington Park Cemetery in St. Louis, Missouri, and a European American rib sample composed of cadavers, autopsies, and forensic cases from Missouri reveals the existence of complex age-associated patterns for differences in measures of intracortical remodeling and cortical area. Females from the two samples express similar bone dimensions and dynamics. The African American females appear to lose more bone than their male counterparts, but this difference is absent in the European American sample. When age-associated patterns are considered, it is in the younger cohorts that African Americans exhibit greater relative cortical area than European Americans, but this is reversed in the older ages, when the latter group manifests greater bone mass. The European American males consistently differ in the slopes and intercepts for the variables compared to the other groups, and differences are highly significant with African American females, with the former group maintaining bone mass while the latter exhibit a more rapid bone loss. Achieving larger relative cortical area due to smaller endosteal area, coupled with better bone quality due to lower intracortical porosity early in life, may be a mechanism by which African Americans, especially females, maintain adequate bone mass in older ages, which buffers them from bone loss and related fragility fractures despite higher rates of intracortical remodeling and endosteal expansion later in life. These results suggest that both genetic and environmental factors are responsible for the differences in bone remodeling and bone mass observed between these samples.     

A regulated nucleocytoplasmic shuttle contributes to Bright's function as a transcriptional activator of immunoglobulin genes

Bright/ARID3a has been implicated in mitogen- and growth factor-induced up-regulation of immunoglobulin heavy-chain (IgH) genes and in E2F1-dependent G1/S cell cycle progression. For IgH transactivation, Bright binds to nuclear matrix association regions upstream of certain variable region promoters and flanking the IgH intronic enhancer. While Bright protein was previously shown to reside within the nuclear matrix, we show here that a significant amount of Bright resides in the cytoplasm of normal and transformed B cells. Leptomycin B, chromosome region maintenance 1 (CRM1) overexpression, and heterokaryon experiments indicate that Bright actively shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. We mapped the functional nuclear localization signal to the N-terminal region of REKLES, a domain conserved within ARID3 paralogues. Residues within the C terminus of REKLES contain its nuclear export signal, whose regulation is primarily responsible for Bright shuttling. Growth factor depletion and cell synchronization experiments indicated that Bright shuttling during S phase of the cell cycle leads to an increase in its nuclear abundance. Finally, we show that shuttle-incompetent Bright point mutants, even if sequestered within the nucleus, are incapable of transactivating an IgH reporter gene. Therefore, regulation of Bright's cellular localization appears to be required for its function.     

Understanding the mechanism of beta-hairpin folding via phi-value analysis

The folding kinetics of a 16-residue beta-hairpin (trpzip4) and five mutants were studied by a laser-induced temperature-jump infrared method. Our results indicate that mutations which affect the strength of the hydrophobic cluster lead to a decrease in the thermal stability of the beta-hairpin, as a result of increased unfolding rates. For example, the W45Y mutant has a phi-value of approximately zero, implying a folding transition state in which the native contacts involving Trp45 are not yet formed. On the other hand, mutations in the turn or loop region mostly affect the folding rate. In particular, replacing Asp46 with Ala leads to a decrease in the folding rate by roughly 9 times. Accordingly, the phi-value for D46A is determined to be approximately 0.77, suggesting that this residue plays a key role in stabilizing the folding transition state. This is most likely due to the fact that the main chain and side chain of Asp46 form a characteristic hydrogen bond network with other residues in the turn region. Taken together, these results support the folding mechanism we proposed before, which suggests that the turn formation is the rate-limiting step in beta-hairpin folding and, consequently, a stronger turn-promoting sequence increases the stability of a beta-hairpin primarily by increasing its folding rate, whereas a stronger hydrophobic cluster increases the stability of a beta-hairpin primarily by decreasing its unfolding rate. In addition, we have examined the compactness of the thermally denatured and urea-denatured states of another 16-residue beta-hairpin, using the method of fluorescence resonance energy transfer. Our results show that the thermally denatured state of this beta-hairpin is significantly more compact than the urea-denatured state, suggesting that the very first step in beta-hairpin folding, when initiated from an extended conformation, probably corresponds to a process of hydrophobic collapse.     

Crystallization and preliminary X-ray studies of an aspartate aminotransferase mutant from Escherichia coli

Mutant aspartate aminotransferase V39L (Val39 replaced by Leu) from Escherichia coli has been crystallized into a monoclinic cell from a polyethylene glycol solution (pH 7.5) by vapor diffusion. The space group and the unit cell dimensions have been determined using a precession camera, a CAD4 diffractometer and a Nicolet Xentronics area detector to be P2(1) with a = 86.8 A, b = 79.9 A, c = 89.4 A, beta = 118.74 degrees. The crystals diffract to better than 2.3 A and are suitable for X-ray structure analysis.