Evaluation of a novel methacrylate‐based protein a resin for the purification of immunoglobulins and Fc‐fusion proteins

Protein A affinity chromatography is a central part of most commercial monoclonal antibody and Fc‐fusion protein purification processes. In the last couple years an increasing number of new Protein A technologies have emerged. One of these new Protein A technologies consists of a novel, alkaline‐tolerant, Protein A ligand coupled to a macroporous polymethacrylate base matrix that has been optimized for immunoglobulin (Ig) G capture. The resin is interesting from a technology perspective because the particle size and pore distribution of the base beads are reported to have been optimized for high IgG binding and fast mass transfer, while the Protein A ligand has been engineered for enhanced alkaline tolerance. This resin was subjected to a number of technical studies including evaluating dynamic and static binding capacities, alkaline stability, Protein A leachate propensity, impurity clearance, and pressure–flow behavior. The results demonstrated similar static binding capacities as those achieved with industry standard agarose Protein A resins, but marginally lower dynamic binding capacities. Removal of impurities from the process stream, particularly host cell proteins, was molecule dependent, but in most instances matched the performance of the agarose resins. This resin was stable in 0.1 M NaOH for at least 100 h with little loss in binding capacity, with Protein A ligand leakage levels comparable to values for the agarose resins. Pressure–flow experiments in lab‐scale chromatography columns demonstrated minimal resin compression at typical manufacturing flow rates. Prediction of resin compression in manufacturing scale columns did not suggest any pressure limitations upon scale up. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1125–1136, 2014     

13C and 1H NMR studies of ionizations and hydrogen bonding in chymotrypsin-glyoxal inhibitor complexes

Benzyloxycarbonyl (Z)-Ala-Pro-Phe-glyoxal and Z-Ala-Ala-Phe-glyoxal have both been shown to be inhibitors of alpha-chymotrypsin with minimal Ki values of 19 and 344 nM, respectively, at neutral pH. These Ki values increased at low and high pH with pKa values of approximately 4.0 and approximately 10.5, respectively. By using surface plasmon resonance, we show that the apparent association rate constant for Z-Ala-Pro-Phe-glyoxal is much lower than the value expected for a diffusion-controlled reaction. 13C NMR has been used to show that at low pH the glyoxal keto carbon is sp3-hybridized with a chemical shift of approximately 100.7 ppm and that the aldehyde carbon is hydrated with a chemical shift of approximately 91.6 ppm. The signal at approximately 100.7 ppm is assigned to the hemiketal formed between the hydroxy group of serine 195 and the keto carbon of the glyoxal. In a slow exchange process controlled by a pKa of approximately 4.5, the aldehyde carbon dehydrates to give a signal at approximately 205.5 ppm and the hemiketal forms an oxyanion at approximately 107.0 ppm. At higher pH, the re-hydration of the glyoxal aldehyde carbon leads to the signal at 107 ppm being replaced by a signal at 104 ppm (pKa approximately 9.2). On binding either Z-Ala-Pro-Phe-glyoxal or Z-Ala-Ala-Phe-glyoxal to alpha-chymotrypsin at 4 and 25 degrees C, 1H NMR is used to show that the binding of these glyoxal inhibitors raises the pKa value of the imidazolium ion of histidine 57 to a value of >11 at both 4 and 25 degrees C. We discuss the mechanistic significance of these results, and we propose that it is ligand binding that raises the pKa value of the imidazolium ring of histidine 57 allowing it to enhance the nucleophilicity of the hydroxy group of the active site serine 195 and lower the pKa value of the oxyanion forming a zwitterionic tetrahedral intermediate during catalysis.     

Expression of DNAJB12 or DNAJB14 Causes Coordinate Invasion of the Nucleus by Membranes Associated with a Novel Nuclear Pore Structure

DNAJB12 and DNAJB14 are transmembrane proteins in the endoplasmic reticulum (ER) that serve as co-chaperones for Hsc70/Hsp70 heat shock proteins. We demonstrate that over-expression of DNAJB12 or DNAJB14 causes the formation of elaborate membranous structures within cell nuclei, which we designate DJANGOS for DNAJ-associated nuclear globular structures. DJANGOS contain DNAJB12, DNAJB14, Hsc70 and markers of the ER lumen and ER and nuclear membranes. Strikingly, they are evenly distributed underneath the nuclear envelope and are of uniform size in any one nucleus. DJANGOS are composed primarily of single-walled membrane tubes and sheets that connect to the nuclear envelope via a unique configuration of membranes, in which the nuclear pore complex appears anchored exclusively to the outer nuclear membrane, allowing both the inner and outer nuclear membranes to flow past the circumference of the nuclear pore complex into the nucleus. DJANGOS break down rapidly during cell division and reform synchronously in the daughter cell nuclei, demonstrating that they are dynamic structures that undergo coordinate formation and dissolution. Genetic studies showed that the chaperone activity of DNAJ/Hsc70 is required for the formation of DJANGOS. Further analysis of these structures will provide insight into nuclear pore formation and function, activities of molecular chaperones, and mechanisms that maintain membrane identity.     

Cyr61 and CTGF are molecular markers of bladder wall remodeling after outlet obstruction

Cysteine-rich protein (Cyr61) and connective tissue growth factor (CTGF) are key immediate early growth factors with functions in cell proliferation, differentiation, and extracellular matrix synthesis. Studies were performed to assess the gene expression profile of Cyr61 and CTGF in rat urinary bladder during growth in response to partial outlet obstruction. The mRNA levels of Cyr61 as determined by ribonuclease protection assay increased sharply after 1 day and remained elevated throughout the time period of the obstruction. This correlates well with increased bladder weight. The CTGF mRNA levels seemed to peak within the second week of the urethral obstruction and correlate well with increased type I collagen mRNA. The expression pattern of either Cyr61 or CTGF proteins corroborated that of their respective mRNAs. Immunohistochemical analyses showed that immunoreactivity of Cyr61 was confined to detrusor smooth muscle and that of CTGF was detected within both detrusor muscle and lamina propria layers. These data strongly indicate the involvement of Cyr61 and CTGF in bladder wall remodeling as a result of the outlet obstruction.     

West African and Amerindian ancestry and risk of myocardial infarction and metabolic syndrome in the Central Valley population of Costa Rica

Genetic ancestry and environmental factors may contribute to the ethnic differences in risk of coronary heart disease (CHD), metabolic syndrome (MS) or its individual components. The population of the Central Valley of Costa Rica offers a unique opportunity to assess the role of genetic ancestry in these chronic diseases because it derived from the admixture of a relatively small number of founders of Southern European, Amerindian, and West African origin. We aimed to determine whether genetic ancestry is associated with risk of myocardial infarction (MI), MS and its individual components in the Central Valley of Costa Rica. We genotyped 39 ancestral informative markers in cases (n = 1,998) with a first non-fatal acute MI and population-based controls (n = 1,998) matched for age, sex, and area of residence, to estimate individual ancestry proportions. Odds ratios (ORs) and 95% confidence intervals (95% CI) were estimated using conditional (MI) and unconditional (MS and its components) logistic regression adjusting for relevant confounders. Mean individual ancestry proportions in cases and controls were 57.5 versus 57.8% for the Southern European, 38.4 versus 38.3% for the Amerindian and 4.1 versus 3.8% for the West African ancestry. Compared with Southern European ancestry, each 10% increase in West African ancestry was associated with a 29% increase in MI, OR (95% CI) = 1.29 (1.07, 1.56), and with a 30% increase on the risk of hypertension, OR (95% CI) = 1.30 (1.00, 1.70). Each 10% increase in Amerindian ancestry was associated with a 14% increase on the risk of MS, OR (95% CI) = 1.14 (1.00, 1.30), and 20% increase on the risk of impaired fasting glucose, OR (95% CI) = 1.20 (1.01, 1.42). These results show that the high variability of admixture proportions in the Central Valley population offers a unique opportunity to uncover the genetic basis of ethnic differences on the risk of disease.     

Different effects of concentric and eccentric muscle actions on plasma volume

The effects of concentric (CON) and eccentric (ECC) contractions on Delta plasma volume (PV), heart rate (HR), and lactate in responses to protocols in different body positions were investigated. CON or ECC contractions were performed in either a single-exercise (6 sets of 12 repetitions of leg extensions completed at 80% of 12 repetition maximum [12RM] with 3-minute rest periods) or multiexercise (4 sets of 10 repetitions for both CON and ECC trials of bench press, leg extension, military press, and leg curl at 80% of 10RM with 90-second rest periods) protocols. HR and lactate increased significantly for both protocols from pre- to postexercise for CON but not ECC trials. DeltaPV was greater following both CON single-exercise (-11.48 +/- 1.38%) and multiexercise (-4.64 +/- 0.33%) trials vs. ECC single-exercise (-1.62 +/- 1.69%) and multiexercise (-1.26 +/- 1.20) trials. Data demonstrate ECC exercise in response to single and multiexercise protocols at the same absolute workload as CON exercise produces less cardiovascular stress.     

Mean residence time of O horizon carbon along a climatic gradient in Scandinavia estimated by 14C measurements of archived soils

We used two datasets of ¹⁴C analyses of archived soil samples to study carbon turnover in O horizons from spruce dominated old-growth stands on well-drained podzols in Scandinavia. The main data set was obtained from archived samples from the National Forest Soil Inventory in Sweden and represents a climatic gradient in temperature. Composite samples from 1966, 1972, 1983 and 2000 from four different regions in a latitude gradient ranging from 57 to 67°N were analysed for ¹⁴C content. Along this gradient the C stock in the O horizon ranges from 2.1 kg m^?2 in the north to 3.7 kg m^?2 in the southwest. The other data set contains ¹⁴C analyses from 1986, 1987, 1991, 1996 and 2004 from the O horizons in Birkenes, Norway. Mean residence times (MRT) were calculated using a two compartment model, with a litter decomposition compartment using mass loss data from the literature for the three-first years of decomposition and a humus decomposition compartment with a fitted constant turnover rate. We hypothesized that the climatic gradient would result in different C turnover in different parts of the country between northern and southern Sweden. The use of archived soil samples was very valuable for constraining the MRT calculations, which showed that there were differences between the regions. Longest MRT was found in the northernmost region (41 years), with decreasing residence times through the middle (36 years) and central Sweden (28 years), then again increasing in the southwestern region (40 years). The size of the soil organic carbon (SOC) pool in the O horizon was mainly related to differences in litter input and to a lesser degree to MRT. Because N deposition leads both to larger litter input and to longer MRT, we suggest that N deposition contributes significantly to the latitudinal SOC gradient in Scandinavia, with approximately twice as much SOC in the O horizon in the south compared to the north. The data from Birkenes was in good agreement with the Swedish dataset with MRT estimated to 34 years.     

Nonaccidental properties underlie shape recognition in Mammalian and nonmammalian vision

An infinite number of 2D patterns on the retina can correspond to a single 3D object. How do visual systems resolve this ill-posed problem and recognize objects from only a few 2D retinal projections in varied exposure conditions? Theories of object recognition rely on the nonaccidental statistics of edge properties, mainly symmetry, collinearity, curvilinearity, and cotermination. These statistics are determined by the image-formation process (i.e., the 2D retinal projection of a 3D object ); their existence under a range of viewpoints enables viewpoint-invariant recognition. An important question in behavioral biology is whether the visual systems of nonmammalian animals have also evolved biases to utilize nonaccidental statistics . Here, we trained humans and pigeons to recognize four shapes. With the Bubbles technique, we determined which stimulus properties both species used to recognize the shapes. Both humans and pigeons used cotermination, the most diagnostic nonaccidental property of real-world objects, despite evidence from a model computer observer that cotermination was not the most diagnostic pictorial information in this particular task. This result reveals that a nonmammalian visual system that is different anatomically from the human visual system is also biased to recognize objects from nonaccidental statistics.