Boronic acids for affinity chromatography: spectral methods for determinations of ionization and diol-binding constants

Arylboronic acids attached to solid matrices have proved useful for the diol-specific chromatography of biomolecules and affinity purification of enzymes by exchangeable-ligand chromatography. The latter use has been limited by the intrinsic ionization constant (pKa approximately 9) of the most common commercial products. The synthesis of several arylboronic acids with ionization constants near neutrality are described, and the application of a new general, spectral-difference method for determining acid ionization constants and formation constants with fructose is developed. In particular 4-(N-methyl) carboxamido-benzeneboronic acid was found to have a pKa of 7.86 and a formation constant with D-fructose of 8600. It was stable toward acid or base hydrolysis. We suggest that 4-carboxybenzeneboronic acid might be useful for preparing matrices for enzyme affinity chromatography.     

Correlations among leaf CO2-exchange rates,areas and enzyme activities among soybean cultivars

Soybean (Glycine max (L.) Merr.) genotypes varying in area per nodal unit (usually a trifoliolate) and maturity class were grown in plots at the University of Illinois experimental farm. Leaf CO₂-exchange rates per unit area (CER) were measured under sunlight on intact plants. In addition to previously reported correlations with specific leaf weight and chlorophyll, CER was positively correlated with ribulose bisphosphate carboxylase (RuBPcase) activity, specific activity, and soluble protein, and was negatively correlated with area per leaf unit. The CER: chlorophyll correlation was destroyed by high CER values in 2 chlorophyll-deficient lines. CER values for 27 of the 35 lines tested fell within the range of those for isolines of cultivar Clark varying in leaf characteristics. The CER values were highest for fully expanded leaves during rapid pod fill. These results suggested that photoperiod (maturity) genes and genes for leaf area growth interact with genes controlling photosynthetic CO₂-exchange to produce the major differences in CER values among soybean genotypes.     

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

A major downside of double-strand break-mediated genome editing is the need to verify the genomic integrity of the targeted locus and confirm the absence of off-target indels. This study shows that in-trans paired nicking is a mutation-free CRISPR strategy to introduce precise knock-ins into human organoids; its genomic fidelity allows all knock-in cells to be pooled, accelerating the establishment of new organoid models.     

Bovine Milk Proteome in the First 9 Days: Protein Interactions in Maturation of the Immune and Digestive System of the Newborn

In order to better understand the milk proteome and its changes from colostrum to mature milk, samples taken at seven time points in the first 9 days from 4 individual cows were analyzed using proteomic techniques. Both the similarity in changes from day 0 to day 9 in the quantitative milk proteome, and the differences in specific protein abundance, were observed among four cows. One third of the quantified proteins showed a significant decrease in concentration over the first 9 days after calving, especially in the immune proteins (as much as 40 fold). Three relative high abundant enzymes (XDH, LPL, and RNASE1) and cell division and proliferation protein (CREG1) may be involved in the maturation of the gastro-intestinal tract. In addition, high correlations between proteins involved in complement and blood coagulation cascades illustrates the complex nature of biological interrelationships between milk proteins. The linear decrease of protease inhibitors and proteins involved in innate and adaptive immune system implies a protective role for protease inhibitor against degradation. In conclusion, the results found in this study not only improve our understanding of the role of colostrum in both host defense and development of the newborn calf but also provides guidance for the improvement of infant formula through better understanding of the complex interactions between milk proteins.     

Grain Protein Accumulation and the Relationship between Leaf Nitrate Reductase and Protease Activities during Grain Development in Maize (Zea mays L.): I. VARIATION BETWEEN GENOTYPES

Four maize hybrids, two with high and two with low levels of postanthesis nitrate reductase activity were grown under field conditions. The characteristic enzyme patterns had been established in previous work. Nitrate reductase and proteases were measured in three representative leaves (ear leaf, fourth leaf above and fourth leaf below the ear) at intervals throughout the period of grain development. Concurrent with enzyme sampling, other plants were harvested and subdivided into top, middle and lower leaves, husks, stalks, and ear. Dry weights, nitrate, and reduced N were determined on all plant parts for each sampling. These data established the rate of N accumulation by the grain and depletion from the vegetative material and provide some insight into the relation between newly reduced and remobilized N and accumulation of grain N. Other plants were harvested at maturity for yield and harvest indices for dry weight and N.     

The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities

Humans contain many HSP (heat-shock protein) 70/HSPA- and HSP40/DNAJ-encoding genes and most of the corresponding proteins are localized in the cytosol. To test for possible functional differences and/or substrate specificity, we assessed the effect of overexpression of each of these HSPs on refolding of heat-denatured luciferase and on the suppression of aggregation of a non-foldable polyQ (polyglutamine)-expanded Huntingtin fragment. Overexpressed chaperones that suppressed polyQ aggregation were found not to be able to stimulate luciferase refolding. Inversely, chaperones that supported luciferase refolding were poor suppressors of polyQ aggregation. This was not related to client specificity itself, as the polyQ aggregation inhibitors often also suppressed heat-induced aggregation of luciferase. Surprisingly, the exclusively heat-inducible HSPA6 lacks both luciferase refolding and polyQ aggregation-suppressing activities. Furthermore, whereas overexpression of HSPA1A protected cells from heat-induced cell death, overexpression of HSPA6 did not. Inversely, siRNA (small interfering RNA)-mediated blocking of HSPA6 did not impair the development of heat-induced thermotolerance. Yet, HSPA6 has a functional substrate-binding domain and possesses intrinsic ATPase activity that is as high as that of the canonical HSPA1A when stimulated by J-proteins. In vitro data suggest that this may be relevant to substrate specificity, as purified HSPA6 could not chaperone heat-unfolded luciferase but was able to assist in reactivation of heat-unfolded p53. So, even within the highly sequence-conserved HSPA family, functional differentiation is larger than expected, with HSPA6 being an extreme example that may have evolved to maintain specific critical functions under conditions of severe stress.     

Availability of reduced N and carbohydrates for ear development of maize

Changes in dry weights, reduced N, nitrate, and nitrate reductase activity of various plant parts of the above ground vegetation (stover) and ears of field grown maize were measured at intervals between anthesis and grain maturity. Nonstructural carbohydrate contents were also measured in some instances. Changes in dry weight and reduced N content were used to approximate net in situ photosynthetic and nitrate assimilation activities and to determine whether the availability of photosynthate or reduced N was limiting grain production.