Are molecular weights of proteins determined by superose 12 column chromatography correct?

Our research on several proteins indicates that accurate molecular weights cannot be determined by Superose 12 column chromatography. In support of this statement, we present data on molecular weights of purified red kidney bean alpha-amylase inhibitor (RKB alphaAI) and white kidney bean alpha-amylase inhibitor (WKB alphaAI) to document this problem. The molecular weight of purified RKB alphaAI determined by Sephadex G-100 gel filtration, polyacrylamide gel electrophoresis, Superose 12 gel filtration and cDNA were 49.0, 51.0, 22.9, and 49.805 kDa (not glycosylated), respectively. The molecular weights of WKB alphaAI by several methods were as follows: Sephadex G-100 gel filtration, 51.0 kDa; Superose 12 gel filtration in 0.2 M NaCl buffer, 23.1 kDa; polyacrylamide gel electrophoresis (PAGE), 51.0 kDa; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 45.0 kDa; multiangle laser light scattering (MALLS), 49.940 kDa; laser-assisted time-of-flight mass spectrometry (LATOFMS), 56.714 kDa; and cDNA sequence (with 12.2% carbohydrate), 55.9 kDa. The data indicate there is ionic interaction between proteins and the matrix of Superose 12 in low ionic strength buffers and hydrophobic interaction at higher ionic strength buffers. Researchers should be cautious when using Superose 12 columns for molecular weight determinations.     

Methionine-containing proteins in twoPhaseolus vulgaris cultivars with different methionine bioavailabilities

The full utilization of legumes as human food is limited by a deficiency of sulfur amino acids, low protein digestibility, low methionine bioavailability and the presence of anti-nutritional factors. A new cultivar ofPhaseolus vulgaris (Carioca 80) has 56.8% available methionine, compared with 29.3% availability in the parent cultivar Carioca. The total methionine content, denaturing gel electrophoretic patterns of methionine-containing proteins, and the percentage of phaseolins (the major storage proteins in Phaseolus) relative to the total protein are similar in the two cultivars. Although the digestibility of the two cultivars is similar, the increased biological value of Carioca 80 may indicate that there are differences in overall bean composition that affect protein hydrolysis and utilization. We suggest the tentative explanation that this is due to differences in the distribution of methionine in the methionine-containing proteins of the two cultivars.     

Biochemical characterization of cystine lyase from broccoli (Brassica oleracea var. italica)

Cystine lyase is the enzyme responsible for off-aroma deterioration in fresh unblanched broccoli. In this research, cystine lyase purification from broccoli has been optimized. Only one protein peak with cystine lyase activity was found during purification. Broccoli cystine lyase was purified 100-fold to homogeneity. L-Cystine, L-cysteine-S-sulfate, L-djenkolic acid, and some S-alkyl-L-cysteines and their sulfoxides are substrates, but the enzyme had negligible activity with L-cystathionine. A K(m) value of 81.2 microM was found for L-cystine. Inhibition and K(i) determinations indicated that L-cysteine is a linear noncompetitive inhibitor with a K(i) of 5 mM and DL-homocysteine is a competitive inhibitor with a K(i) of 1.5 mM. The molecular weight of cystine lyase was determined to be 100 kDa by three methods, with two subunits of 48 kDa each and a carbohydrate content of 3%. Further characterization included cofactor quantification, the effects of temperature and pH on activity and stability, and amino acid composition.     

Impacting Librarianship and Veterinary Medicine: History of the Veterinary Medical Libraries Section of the Medical Library Association from 1974 to 2014

This article discusses the Veterinary Medical Libraries Section (VMLS) of the Medical Library Association (MLA), its impacts on both librarianship and veterinary medicine during its 40-year history, how it achieved those impacts, relationships, selected projects, and organizational changes through the years. A listing of publications about veterinary librarianship and VMLS products is available on the VMLS website (Veterinary Medical Libraries Section, 2014).     

Evaluation of the Physical–Chemical Characteristics of Wastewater After Disinfection with Peracetic Acid

Water, Air, &; Soil Pollution - The use of peracetic acid (PAA) in the disinfection of sanitary effluents has been proposed by various authors. However, there are still doubts about its...     

Adding Zooplankton to the OSMAC Toolkit: Effect of Grazing Stress on the Metabolic Profile and Bioactivity of a Diatom

“One strain many compounds” (OSMAC) based approaches have been widely used in the search for bioactive compounds. Introducing stress factors like nutrient limitation, UV-light or cocultivation with competing organisms has successfully been used in prokaryote cultivation. It is known that diatom physiology is affected by changed cultivation conditions such as temperature, nutrient concentration and light conditions. Cocultivation, though, is less explored. Hence, we wanted to investigate whether grazing pressure can affect the metabolome of the marine diatom Porosira glacialis, and if the stress reaction could be detected as changes in bioactivity. P. glacialis cultures were mass cultivated in large volume bioreactor (6000 L), first as a monoculture and then as a coculture with live zooplankton. Extracts of the diatom biomass were screened in a selection of bioactivity assays: inhibition of biofilm formation, antibacterial and cell viability assay on human cells. Bioactivity was found in all bioassays performed. The viability assay towards normal lung fibroblasts revealed that P. glacialis had higher bioactivity when cocultivated with zooplankton than in monoculture. Cocultivation with diatoms had no noticeable effect on the activity against biofilm formation or bacterial growth. The metabolic profiles were analyzed showing the differences in diatom metabolomes between the two culture conditions. The experiment demonstrates that grazing stress affects the biochemistry of P. glacialis and thus represents a potential tool in the OSMAC toolkit.     

Mitogen-induced lymphocyte proliferation in loggerhead sea turtles: comparison of methods and effects of gender, plasma testosterone concentration, and body condition on immunity

A fully functioning immune system is vital to the survival of threatened and endangered sea turtles. Immunological protection against diseases in any organism can be reduced by a number of natural and anthropogenic factors, such as seasonal changes, malnutrition, disease states, and contaminant exposure. These factors are even more critical when they occur in endangered species or populations. To identify alterations in the immunological health of loggerhead sea turtles (Caretta caretta), the mitogen-induced lymphocyte proliferation (LP) assay was developed using peripheral blood leukocytes (PBLs). Collection and culture conditions were optimized for this assay using non-lethal blood samples collected from free-ranging turtles along the southeastern US coast. During the collection, two anticoagulants (sodium heparin and lithium heparin) were compared to determine effects of different ions on assay results. Optimal culture conditions were established for loggerhead PBLs while two different methods of measuring LP were compared: (1) the traditional radioactive (3)H-thymidine assay and (2) a non-radioactive, colorimetric method utilizing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium (MTT). The results indicate that the (3)H-thymidine and the non-radioactive MTT methods did not correlate with each other and that the use of heparin type did not influence the results of the LP assay. Lastly, using these optimized methods, we investigated the effect of gender, plasma testosterone concentration, and body condition on LP in loggerhead turtles and found that none of the parameters largely influenced LP.     

The use of polymerase chain reaction assay to diagnose proliferative gill disease in channel catfish (Ictalurus punctatus).

To assess the potential of a polymerase chain reaction (PCR) assay as a diagnostic tool in the detection of proliferative gill disease (PGD) in channel catfish (Ictalurus punctatus), PCR assays were compared with the traditional diagnostic methods of gill wet mounts and histology. A PCR assay using primers for Aurantiactinomyxon ictaluri, the actinospore associated with PGD, was performed with tissues from fish from commercial ponds. Using histology as the "gold standard," the sensitivity, specificity, and accuracy of the PCR assay were all >90%. In comparison, the wet mount examinations had a lower sensitivity and specificity. Using the chi-square test and a test for strength of association, there was a significant, strong association between results obtained by PCR and those obtained by the other 2 methods. These results demonstrate that the PCR assay is a good diagnostic tool for the detection of PGD.