A Seed Storage Protein with Possible Self-Affinity through Lectin-Like Binding

The primary storage protein of oat (Avena sativa L.) seeds, globulin, was shown to have a specific carbohydrate-binding activity. The globulin was capable of hemagglutinating rabbit red blood cells and this hemagglutination was inhibited by the β-glucan, laminarin, as well as by carbohydrate which had been cleaved from the native globulin. Globulin with carbohydrate-binding activity was isolated from cell wall preparations and from defatted flour. The lectin activity apparently resides in the α-subunit of the globulin and has affinity for the carbohydrate which is O-glycosidically linked to the globulin. A portion of this carbohydrate is attached to the β-subunit. Two affinity columns were synthesized utilizing laminarin and the carbohydrate from the native globulin as ligands. The hemagglutinating activity bound to both of these columns. The activity was specifically eluted from the globulin-carbohydrate affinity column with carbohydrate cleaved from native globulin by an alkali-catalyzed β-elimination. The possible roles of this unique self-binding capacity are discussed.     

Silver-decorated orthorhombic nanotubes of lithium vanadium oxide: an impeder of bacterial growth and biofilm

Reoccurrence of infectious diseases and ability of pathogens to resist antibacterial action has raised enormous challenges which may possibly be confronted by nanotechnology routes. In the present study, uniformly embedded silver nanoparticles in orthorhombic nanotubes of lithium vanadium oxide (LiV₂O_5/Ag) were explored as an impeder of bacterial growth and biofilm. The LiV₂O₅/Ag nanocomposites have impeded growth of Gram-positive Bacillus subtilis NCIM 2063 and Gram-negative Escherichia coli NCIM 2931 at 60 to 120 μg/mL. It also impeded the biofilm in Pseudomonas aeruginosa NCIM 2948 at 12.5 to 25 μg/mL. Impedance in the growth and biofilm occurs primarily by direct action of the nanocomposites on the cell surfaces of test organisms as revealed by surface perturbation in scanning electron microscopy. As the metabolic growth and biofilm formation phenomena of pathogens play a central role in progression of pathogenesis, LiV₂O₅/Ag nanocomposite-based approach is likely to curb the menace of reoccurrence of infectious diseases. Thus, LiV₂O₅/Ag nanocomposites can be viewed as a promising candidate in biofabrication of biomedical materials.     

A single-step chromatographic method for the purification of proteins devoid of exposed histidine residues

The principle of the immobilized metal affinity chromatography (IMAC) is based on the differences in the affinity of proteins for metal ions bound in a 1:1 complex of iminodiacetic acid (IDA) immobilized on a chromatographic support. A single step purification was carried out for luteinizing hormone (LH) on Cu2+, Zn2+, Ni2+, and Co2+ IDA Sepharose affinity columns. Highly purified LH was obtained with a Cu2+ IDA Sepharose column. SDS-PAGE and Western blot analysis were done to confirm the purity of the hormone. Biological activity has been evaluated by radio-immunoassay. This method was found economically viable and suitable for the recovery of biologically active hormone.     

Analysis of N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1 by a proteomic approach

Pirellula sp. strain 1 is a marine bacterium that can grow with the chitin monomer N-acetylglucosamine as sole source of carbon and nitrogen under aerobic conditions, and that is a member of the bacterial phylum Planctomycetes. As a basis for the proteomic studies we quantified growth of strain 1 with N-acetylglucosamine and glucose, revealing doubling times of 14 and 10 h, respectively. Studies with dense cell suspensions indicated that the capacity to degrade N-acetylglucosamine and glucose may not be tightly regulated. Proteins from soluble extracts prepared from exponential cultures grown either with N-acetylglucosamine or glucose were separated by two-dimensional gel electrophoresis and visualized by fluorescence staining (Sypro Ruby). Analysis of the protein patterns revealed the presence of several protein spots only detectable in soluble extracts of N-acetylglucosamine grown cells. Determination of amino acid sequences and peptide mass fingerprints from tryptic fragments of the most abundant one of these spots allowed the identification of the coding gene on the genomic sequence of Pirellula sp. strain 1. This gene showed similarities to a dehydrogenase from Bacillus subtilis, and is closely located to a gene similar to glucosamine-6-phosphate isomerase from B. subtilis. Genes of two other proteins expressed during growth on N-acetylglucosamine as well as on glucose were also identified and found to be similar to a glyceraldehyde-3-phosphate-dehydrogenase and a NADH-dehydrogenase, respectively. Thus the coding genes of three proteins expressed during growth of Pirellula sp. strain 1 on carbohydrates were identified and related by sequence similarity to carbohydrate metabolism.     

Influence of castration-induced testosterone and estradiol deficiency on obesity and glucose metabolism in male Göttingen minipigs

Low testosterone and estradiol concentrations are predictive for the development of the metabolic syndrome in men and women, respectively. The aim of this study was to investigate the influence of sex hormone deficiency on food intake, body weight, body composition and glucose metabolism in male Göttingen minipigs.Five adult male Göttingen minipigs were studied before castration (pre-cast), 10-18 days (post-cast 1) and 10-11 weeks (post-cast 2) after castration. Parameters of interest were food intake, body weight, body fat percentage and sex hormone concentrations. Furthermore glucose tolerance, glucagon suppression, insulin resistance, beta cell function and disposition index were evaluated by oral and intravenous glucose tolerance tests.Castration led to almost complete disappearance of circulating testosterone and estradiol and secondarily to increased food intake, body weight and body fat percentage. Ten-eighteen days sex hormone deficiency (post-cast 1) did not significantly change any of the investigated metabolic parameters compared to pre-cast levels. Ten weeks after castration (post-cast 2) significant insulin resistance, glucose intolerance and hyperglucagonemia was found, and the beta cell function and the disposition index both were decreased.In conclusion, castration-induced sex hormone deficiency in male Göttingen minipigs results in hyperphagia, obesity and disturbed glucose metabolism, which are some of the features typical for the human metabolic syndrome.     

Position sense in the lumbar spine with torso flexion and loading

Proprioception plays an important role in appropriate sensation of spine position, movement, and stability. Previous research has demonstrated that position sense error in the lumbar spine is increased in flexed postures. This study investigated the change in position sense as a function of altered trunk flexion and moment loading independently. Reposition sense of lumbar angle in 17 subjects was assessed. Subjects were trained to assume specified lumbar angles using visual feedback. The ability of the subjects to reproduce this curvature without feedback was then assessed. This procedure was repeated for different torso flexion and moment loading conditions. These measurements demonstrated that position sense error increased significantly with the trunk flexion (40%, p < .05) but did not increase with moment load (p = .13). This increased error with flexion suggests a loss in the ability to appropriately sense and therefore control lumbar posture in flexed tasks. This loss in proprioceptive sense could lead to more variable lifting coordination and a loss in dynamic stability that could increase low back injury risk. This research suggests that it is advisable to avoid work in flexed postures.     

Anthranilate derivatives as TACE inhibitors: Docking based CoMFA and CoMSIA analyses

Anthranilic acid based derivatives (ANTs) have been identified as a novel class of potent tumor necrosis factor-α converting enzyme (TACE) inhibitors. A computational strategy based on molecular docking studies, followed by CoMFA and CoMSIA analyses has been performed to elucidate the atomic details of the TACE/ANT interactions and also to identify the most important features impacting TACE inhibitory activity of ANTs. The CoMSIA model resulted to be slightly more predictive than CoMFA model, and gave conventional r² 0.991, r_cv² 0.793, q² 0.777, SEE 0.050, F-value 655.610, and r_test² 0.871. The 3D-QSAR field contributions and the structural features of the TACE binding site showed a good correlation. These studies will be useful to design new TACE inhibitors with improved potency.     

Imaging of alkaline phosphatase activity in bone tissue

The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP) using a small imaging molecule in combination with (19)Flourine magnetic resonance spectroscopic imaging ((19)FMRSI). 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using (19)Fluorine magnetic resonance spectroscopy ((19)FMRS) and (19)FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. (19)FMRS and (19)FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. (19)FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized (19)FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of (19)FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, (19)FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications.     

Role of proteins in resistance mechanism of Pseudomonas fluorescens against heavy metal induced stress with proteomics approach

The genus Pseudomonas is a group of gram-negative, motile, rod-shaped bacteria known for their metabolic versatility. One of the species is Pseudomonas fluorescens, which has an efficient system for detoxification of industrial waste. Other aspects include, catabolic versatility, excellent root colonizing abilities and capacity to produce a wide range of antifungal metabolites. They are also known for their resistance and survival in the presence of several organic and inorganic pollutants. P. fluorescens has also been isolated from metal polluted water and soils but the elucidation of proteins responsible for its survival is still not clear. The aim of the study was to elucidate the differential protein expression of this bacterium when exposed to heavy metal stress, using two-dimensional electrophoresis. The proteins spo VG and enolase showed upregulation during the bacterial exposure to lead and copper. Hypothetical protein showed downregulation when bacterium was exposed to cobalt. Some proteins like xylosyltransferase, ORF 18 phage phi KZ, OMP H1 and translational elongation factor EF-Tu appeared only during their exposure to cobalt. These were absent in the control condition. Analysis of the differentially expressed proteins as well as the newly synthesized proteins along with the results obtained growth and enzyme activity indicate the involvement of all these factors in the survival of this organism in the presence of heavy metals.     

Effect of rearing system and mixing at loading on transport and lairage behaviour and meat quality: comparison of outdoor and conventionally raised pigs

The effect of raising pigs outdoors or conventionally in a barren environment was investigated with respect to behaviour during a 3 to 4 h journey and a 2 h lairage, blood chemistry at slaughter and meat quality characteristics. Pigs were either kept in farm pen groups or were mixed at loading and kept in the groups, so formed, until slaughter. Non-mixed outdoor pigs settled more quickly during transport and lay down to a greater extent at the end of the journey and lairage period compared with conventionally raised pigs. Mixing led to fewer pigs sitting and lying during transport for conventionally raised pigs, where nearly 80% were still standing at the end of the journey. Mixing had no effect on pig posture in the lairage. Outdoor pigs were less aggressive than conventionally raised pigs especially during lairage and had a lower frequency of unacceptable skin damage in the rear and shoulder area. Aggressive interactions were almost exclusively confined to mixed groups and occurred mainly between pigs from different farm pens, i.e. between unfamiliar animals. Mixing at loading led therefore to higher levels of unacceptable skin damage. Cortisol concentrations in slaughter blood were not affected by rearing system or mixing, but mixed, conventionally raised pigs had higher plasma creatine kinase (CK) activities than non-mixed ones (1132 v. 761 U/l, respectively, P < 0.05). Outdoor pigs had similar CK activities, irrespective of mixing (682 and 771 U/l for mixed and non-mixed, respectively). Muscle pH early post mortem was highest in outdoor pigs and muscle temperature lower, but no pig showed pH values below 6.0. Ultimate pH values were both higher (Semimembranosus (S)) and lower (Semispinalis capitis (SC)) than in conventionally raised pigs, and outdoor pigs tended to have a lower frequency of higher than normal pH values. Internal reflectance (MQM) values in Biceps femoris (BF) were highest in outdoor pigs but the incidence of pale, soft and exudative (PSE) meat was low, varying between 0% and 1% for experimental groups. In general, the effects of rearing system and mixing on meat quality measurements taken early post mortem or the day after slaughter were slight, but the trends seen support the CK results, and show that conventionally raised pigs may have found mixing pre-slaughter to be more physically stressful than outdoor pigs did.