Quantitative analysis of phytate globoids isolated from wheat bran and characterization of their sequential dephosphorylation by wheat phytase

Wheat phytase was purified to investigate the action of the enzyme toward its pure substrate (phytic acid - myo-inositol hexakisphosphate) and its naturally occurring substrate (phytate globoids). Phytate globoids were purified to homogeneity from wheat bran, and their nutritionally relevant parameters were quantified by ICP-MS. The main components of the globoids were phytic acid (40% w/w), protein (46% w/w), and several minerals, in particular, K > Mg > Ca > Fe (in concentration order). Investigation of enzyme kinetics revealed that K(m) and V(max) decreased by 29 and 37%, respectively, when pure phytic acid was replaced with phytate globoids as substrate. Time course degradation of phytic acid or phytate globoids using purified wheat phytase was followed by HPIC identification of inositol phosphates appearing and disappearing as products. In both cases, enzymatic degradation initiated at both the 3- and 6-positions of phytic acid and end products were inositol and phosphate.     

Formation of aroma compounds during long-term frozen storage of unblanched leek (Allium ampeloprasum Var. Bulga) as affected by packaging atmosphere and slice thickness

Content of aroma compounds and catalytic activity of lipoxygenase (LOX), hydroperoxide lyase (HPL), and alcohol dehydrogenase (ADH) were analyzed in 4- and 15-mm unblanched leek slices packed in atmospheric air (4- and 15-mm) or 100% nitrogen (N) (only 15-mm) seven times during 12 months of frozen storage (12M). Total amount of sulfur compounds was influenced by storage time, slice thickness, and atmosphere (concentration in fresh 4-mm slices = 17.8 mg/L, 4-mm 12M = 3.48 mg/L, fresh 15-mm slices = 2.48 mg/L, 15-mm 12M = 0.418 mg/L and 15-mm N 12M = 1.81 mg/L). The 4-mm slices significantly developed the most aldehydes after 12M (total amount = 9.28 mg/L) compared to 15-mm 12M (6.49 mg/L) and 15-mm N 12M (4.33 mg/L). LOX activity is positively influenced by nitrogen packaging, and HPL activity is influenced by slice thickness, whereas ADH is unaffected by both parameters.     

Rolling behaviour of sows in relation to piglet crushing on sloped versus level floor pens

The study focused on the rolling behaviour of sows and the crushing of piglets by sows' rolling behaviour. The experiment examined the influence of sloped floor in loose housed farrowing pens on the rolling behaviour of sows and crushing of piglets. The experimental unit was made up of 24 pens. There were two experimental pen designs with piglet creep in the corner of the pen and piglet creep across the end of the pen, respectively. Both of the experimental pen designs had a 10% sloped floor in the sow's resting area. The two control pen designs were identical to the experimental pen designs, but with a level floor. The behaviour of 85 sows and their litters was continuously video recorded. Behavioural observations were made from birth of the first piglet and until 3 days after birth of the first piglet. Rolling behaviour of sows caused significantly more trapped piglets under the sow than lying down from standing (P = 0.04). Rolling behaviour caused 64% of the trapped piglets and lying down from standing caused 36% of the trapped piglets. Rolling from udder to side without protection trapped significantly more piglets than rolling from udder to side near slanted wall or piglet protection rails and rolling from side to udder (P < 0.001). With a certain pen design sloped floor reduced rolling from udder to side without protection (P = 0.007) and reduced the number of trapped piglets (P = 0.01), but results concerning lying behaviour showed that sloped floor pushed sows to rest on the level part of the floor. The results indicate that rolling behaviour that crushes piglets can be reduced, and sows prefer to lie on a level floor.     

Derivatives of erythropoietin that are tissue protective but not erythropoietic

Erythropoietin (EPO) is both hematopoietic and tissue protective, putatively through interaction with different receptors. We generated receptor subtype-selective ligands allowing the separation of EPO's bioactivities at the cellular level and in animals. Carbamylated EPO (CEPO) or certain EPO mutants did not bind to the classical EPO receptor (EPOR) and did not show any hematopoietic activity in human cell signaling assays or upon chronic dosing in different animal species. Nevertheless, CEPO and various nonhematopoietic mutants were cytoprotective in vitro and conferred neuroprotection against stroke, spinal cord compression, diabetic neuropathy, and experimental autoimmune encephalomyelitis at a potency and efficacy comparable to EPO.     

Characterization of Tolerance Limit in Spirulina platensis in Relation to Nanoparticles

A study was carried out under in vitro conditions to characterize the growth of blue green alga, Spirulina platensis, in standard CFTRI medium containing different nanoparticles of copper oxide (CuO) (50 nm, 10 ppm), zinc oxide (ZnO) (50 nm, 10 ppm), tricalcium phosphate (TCP) (<100 nm, 90 ppm), and hydroxy apatite (HA) (<200 nm, 90 ppm). S. platensis exhibited significant higher growth in standard CFTRI medium containing 90 ppm phosphorus as nanoparticles of TCP and HA. On the other hand, calcium phosphate nanoparticles caused significant reduction in nitrate reductase activity as well as in protein content of the alga. Marked change in chlorophyll-a/b ratio was also noted when phosphorus was supplied through nano tricalcium phosphate and nano hydroxy apatite particles as compared to ionic form (K₂HPO₄). The study revealed that the growth of Spirulina in the presence of ZnO nanoparticles was retarded, while no growth was observed with CuO nanoparticles. It was concluded that alga Spirulina showed much sensitivity to nanoparticles of zinc and copper (<50 nm) and was able to tolerate the toxicity of nanophosphate (tricalcium phosphate <100 nm; hydroxy apatite <200 nm).     

Inhibition of virulence gene expression in Staphylococcus aureus by novel depsipeptides from a marine photobacterium

During a global research expedition, more than five hundred marine bacterial strains capable of inhibiting the growth of pathogenic bacteria were collected. The purpose of the present study was to determine if these marine bacteria are also a source of compounds that interfere with the agr quorum sensing system that controls virulence gene expression in Staphylococcus aureus. Using a gene reporter fusion bioassay, we recorded agr interference as enhanced expression of spa, encoding Protein A, concomitantly with reduced expression of hla, encoding α-hemolysin, and rnaIII encoding RNAIII, the effector molecule of agr. A marine Photobacterium produced compounds interfering with agr in S. aureus strain 8325-4, and bioassay-guided fractionation of crude extracts led to the isolation of two novel cyclodepsipeptides, designated solonamide A and B. Northern blot analysis confirmed the agr interfering activity of pure solonamides in both S. aureus strain 8325-4 and the highly virulent, community-acquired strain USA300 (CA-MRSA). To our knowledge, this is the first report of inhibitors of the agr system by a marine bacterium.     

Anthelmintic and antimicrobial activity of methanolic and aqueous extracts of Euphorbia helioscopia L.

The aim of this study was to evaluate the anthelmintic and antimicrobial efficacy of Euphorbia helioscopia crude extracts. A worm motility inhibition assay and egg hatch assay were used for in vitro study, and a faecal egg count reduction assay was used for in vivo study. The in vitro study revealed anthelmintic effects of crude methanolic extracts of E. helioscopia on live Haemonchus contortus worms as evident from their paralysis and/or death at 8 h after exposure. Different concentrations (12.5 mg?ml^?1, 25 mg?ml^?1 and 50 mg?ml^?1) of aqueous and methanolic extracts were used against H. contortus which exhibited dose-dependent anthelmintic effects on H. contortus. Different extracts of E. helioscopia on percent inhibitory egg hatching was very low as compared to levamisole. The antimicrobial activity of extracts ranging from 100 to 500 mg?ml^?1 screened by disc diffusion method against four selected bacterial (Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas multocida and Escherichia coli) and two fungal strains (Aspergillus flavus and Candida albicans) was also dose dependent with the extract showing more inhibitory effects against S. aureus and E. coli and a minimum inhibitory effect against A. flavus. It is concluded that the entire plant of E. helioscopia possesses significant anthelmintic and antimicrobial activity and could be a potential alternative for treating cases of helminth infections in ruminants.     

Assessment of the effects of intramuscular administration of alfaxalone with and without medetomidine in Horsfield's tortoises (Agrionemys horsfieldii)

ObjectiveTo characterise four different intramuscular (IM) anaesthetic protocols, two with alfaxalone and two with alfaxalone in combination with medetomidine in terrestrial tortoises.Study designBlinded, randomized, cross‐over experimental study.AnimalsNine healthy adult male Horsfield's tortoises (Agrionemys horsfieldii).MethodsEach tortoise was randomly assigned to one of four different protocols: 1) 10 mg kg^?1 alfaxalone; 2) 10 mg kg^?1 alfaxalone + 0.10 mg kg^?1 medetomidine; 3) 20 mg kg^?1 alfaxalone; and 4) 20 mg kg^?1 alfaxalone + 0.05 mg kg^?1 medetomidine. During the experiment, the following variables were recorded: heart rate; respiratory rate; peripheral nociceptive responses; muscle strength; ability to intubate; palpebral, corneal and tap reflexes; and cloacal temperature.ResultsProtocols 1 and 2 resulted in moderate sedation with no analgesia, and moderate to deep sedation with minimal analgesia, respectively. Protocols 3 and 4 resulted in deep sedation or anaesthesia with variable analgesic effect; these two protocols had the longest total anaesthetic time and allowed intubation in 6/9 and 8/9 tortoises respectively. The total anaesthesia/sedation time produced by alfaxalone was significantly increased (p <0.05) by the addition of medetomidine. There were no significant differences regarding time to plateau phase and duration of plateau phase. Baseline heart rate of 53 ± 6 beats minute^?1 decreased significantly (p <0.05) with all protocols, and was lower (p <0.05) in protocols 3 and 4. Heart rate increased after atipamezole administration, but the increase was transient. In two tortoises, extreme bradycardia with no cardiac activity for 10 minutes was observed with protocols 3 and 4.Conclusion and clinical relevanceAlfaxalone 10 and 20 mg kg^?1 IM can be used for sedation for non‐painful procedures. Alfaxalone in combination with medetomidine can be used for deeper sedation or anaesthesia, but the observed respiratory and cardiovascular depression may limit its use.     

Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs,pig carcasses and human

Background

Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3) in E. coli isolated from pigs and humans with a specific objective to assess the genetic diversity of plasmids involved in the mobility of sul genes.

Methods

A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their susceptibility to selected antimicrobial. Multiplex PCR was conducted to detect the presence of three sul genes among the sulfonamide-resistant E. coli isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids. Plasmids carrying sul genes were characterized by PCR-based replicon typing to allow a comparison of the types of sul genes, the reservoir and plasmid present.

Results

A total of 109/501 isolates exhibited sulfonamide resistance. The relative prevalences of sul genes from the three reservoirs (pigs, pig carcasses and humans) were 65%, 45% and 12% for sul2, sul1, and sul3, respectively. Transfer of resistance through conjugation was observed in 42/57 isolates. Resistances to streptomycin, ampicillin and trimethoprim were co-transferred in most strains. Class 1 integrons were present in 80% of sul1-carrying plasmids and 100% of sul3-carrying plasmids, but only in 5% of sul2-carrying plasmids. The sul plasmids ranged from 33 to 160-kb in size and belonged to nine different incompatibility (Inc) groups: FII, FIB, I1, FIA, B/O, FIC, N, HI1 and X1. IncFII was the dominant type in sul2-carrying plasmids (52%), while IncI1 was the most common type in sul1 and sul3-carrying plasmids (33% and 45%, respectively). Multireplicons were found associated with all three sul genes.

Conclusions

Sul genes were distributed widely in E. coli isolated from pigs and humans with sul2 being most prevalent. Sul-carrying plasmids belonged to diverse replicon types, but most of detected plasmids were conjugative enabling horizontal transfer. IncFII seems to be the dominant replicon type in sul2-carrying plasmids from all three sources.