One-step purification and immobilization of his-tagged rhamnosidase for naringin hydrolysis

α-l-Rhamnosidase (EC 3.2.1.40) is an enzyme that catalyzes the cleavage of terminal rhamnoside groups from naringin to prunin and rhamnose. In this study, a His-tag was genetically attached to the rhamnosidase gene ramA from Clostridium stercorarium to facilitate its purification from Escherichia coli BL21 (DE3) cells containing the pET-21d/ramA plasmid. Immobilized metal-chelate affinity chromatography (IMAC) resulted in one-step purification of N-terminally His-tagged recombinant rhamnosidase (N-His-CsRamA) which was immobilized in Ca²⁺ alginate (3%) beads. The optimum pH levels of the free and immobilized recombinant rhamnosidase were found to be 6.0 and 7.5, and the optimum temperature 55 and 60 °C respectively. At 50 °C, the free enzyme was relatively stable and exhibited a less than 50% reduction in residual activity after 180 min of incubation. The free and immobilized enzymes achieved 76% and 67% hydrolysis of the naringin in Kinnow juice respectively. Immobilization of recombinant rhamnosidase enabled its reutilization up to 9 hydrolysis batches without an appreciable loss in activity. This result indicated that the His-tagged thermostable rhamnosidase could be prepared as described and may serve to illustrate an economical and commercially viable process for industrial application.

Complex coacervation between flaxseed protein isolate and flaxseed gum

Flaxseed protein isolate (FPI) and flaxseed gum (FG) were extracted, and the electrostatic complexation between these two biopolymers was studied as a function of pH and FPI-to-FG ratio using turbidimetric and electrophoretic mobility (zeta potential) tests. The zeta potential values of FPI, FG, and their mixtures at the FPI-to-FG ratios of 1:1, 3:1, 5:1, 10:1, 15:1 were measured over a pH range 8.0-1.5. The alteration of the secondary structure of FPI as a function of pH was studied using circular dichroism. The proportion of a-helical structure decreased, whereas both β-sheet structure and random coil structure increased with the lowering of pH from 8.0 to 3.0. The acidic pH affected the secondary structure of FPI and the unfolding of helix conformation facilitated the complexation of FPI with FG. The optimum FPI-to-FG ratio for complex coacervation was found to be 3:1. The critical pH values associated with the formation of soluble (pHc) and insoluble (pHΦ1) complexes at the optimum FPI-to-FG ratio were found to be 6.0 and 4.5, respectively. The optimum pH (pHopt) for the optimum complex coacervation was 3.1. The instability and dissolution of FPI-FG complex coacervates started (pHΦ2) at pH2.1. These findings contribute to the development of FPI-FG complex coacervates as delivery vehicles for unstable albeit valuable nutrients such as omega-3 fatty acids.

Synthesis and structure of new oligomethylene-bridged double ladders. How far can the layers be separated?

The synthesis of the α,ω-bis[dichloro(trimethylsilylmethyl)stannyl]alkanes, (Me₃SiCH₂)C1₂Sn(CH₂)_nSnCl₂(CH₂SiMe₃) (13, n=5; 14, n=6; 15, n=7; 16, n=8; 17, n=10; 18, n=12) and the corresponding oligomethylene-bridged diorganotin oxides [(Me₃SiCH₂)(O)Sn(CH₂)_nSn(O)(CH₂SiMe₃)]_m (19, n=5; 20, n=6; 21, n=7; 22, n=8; 23, n=10; 24; n=12) is reported. The reaction of the diorganodichlorostannanes 13–18 with the corresponding diorganotin oxides 19–24 provided the spacer-bridged tetraorganodistannoxanes {[(Me₃SiCH₂)ClSn(CH₂)_nSnCl(CH₂SiMe₃)]O}₄ (25, n=5; 26, n=6; 27, n=7; 28, n=8; 29, n=10; 30, n=12). Compounds 13–30 have been identified by elemental analyses and multinuclear NMR spectroscopy. Compounds 25, 27, 29 and 30 have also been characterised by single crystal X-ray diffraction analysis and electrospray mass spectrometry. For the latter the essential double ladder motif is maintained for all n in the solid state, but subtle changes in alignment of the ladder planes occur. Separation between the two layers of the double ladder ranges from approx. 8.7  Å (for 25, n=5) to approx. 15 Å (for 30, n=12). In solution there is some dissociation of the double ladders into the corresponding dimers. The degree of dissociation is favoured by increasing oligomethylene chain length n.

The power of the 6 three model for enhancing academic teachers' capacities for effective online teaching and learning: benefits, initiatives and future directions

To gain the full educational benefits of the major new investments in corporate technologies supporting online teaching and learning it is argued that a strategic, systems based approach to academic professional development (APD) is required. Such an approach requires a clear view of the key areas of potential and enduring teaching and learning benefit which can be realised from online developments, including an understanding of the changing role of the academic teacher in higher education, the identification of the desired professional capacities to educate online, and the implementation of a number of coordinated initiatives to develop these professional capacities in order to engage constructively with the learning and technology opportunities. Based on previous work, we propose a 6three model of Academic Professional Capacities Development for effective APD of online teaching and learning. The model can help inform the actions of policy makers, executives and practitioners in ways that promote an authentic learning organisation.

Family food environment and dietary behaviors likely to promote fatness in 5-6 year-old children

Background: The family food environment (FFE) is likely to exert important influences on young children's eating. Examination of multiple aspects of the FFE may provide useful insights regarding which of these might most effectively be targeted to prevent childhood obesity.

Objective: To assess the associations between the FFE and a range of obesity-promoting dietary behaviors in 5–6-year-old children.

Design: Cross-sectional study.

Subjects: Five hundred and sixty families sampled from three socio-economically distinct areas.

Measurements: Predictors included parental perceptions of their child's diet, food availability, child feeding practices, parental modeling of eating and food preparation and television (TV) exposure. Dietary outcomes included energy intake, vegetable, sweet snack, savory snack and high-energy (non-dairy) fluid consumption.

Results: Multiple linear regression analyses, adjusted for all other predictor variables and maternal education, showed that several aspects of the FFE were associated with dietary outcomes likely to promote fatness in 5–6-year-old children. For example, increased TV viewing time was associated with increased index of energy intake, increased sweet snack and high-energy drink consumption, and deceased vegetable intake. In addition, parent's increased confidence in the adequacy of their child's diet was associated with increased consumption of sweet and savory snacks and decreased vegetable consumption. 

Conclusion:  This study substantially extends previous research in the area, providing important insights with which to guide family-based obesity prevention strategies.

Overweight and obesity prevalence in children based on 6- or 12- month IOTF cut-points: does interval size matter?

The International Obesity Taskforce (IOTF) recommends using age- and gender-specific body mass index (BMI) cut-points for defining the prevalence of overweight and obesity in children. These are given in both 6- and 12-month age intervals. Since the BMI-for-age curves are nonlinear, a degree of bias will be introduced when age intervals are wide. We aimed to quantify this bias in prevalence estimates in 2178 Australian children aged 4-12 years using 12- versus 6-month age intervals. Using the 12-month interval, the prevalence of overweight and obesity was underestimated by 1.4% compared to the 6-month interval estimates; however, this was age-dependent. It overestimated prevalence for 4-year olds, but underestimated it for older ages by up to 2.6%. Overweight prevalence was generally affected more than obesity prevalence. The use of different age intervals for IOTF cut-points introduces a small but systematic bias in prevalence estimates of overweight and obesity.

Vascular attack by 5,6-dimethylxanthenone-4-acetic acid combined with B7.1 (CD80)-mediated immunotherapy overcomes immune resistance and leads to the eradication of large tumors and multiple tumor foci

The promise of cancer immunotherapy is that it will not only eradicate primary tumors but will generate systemic antitumor immunity capable of destroying distant metastases. A major problem that must first be surmounted relates to the immune resistance of large tumors. Here we reveal that immune resistance can be overcome by combining immunotherapy with a concerted attack on the tumor vasculature. The functionally related antitumor drugs 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone acetic acid (FAA), which cause tumor vasculature collapse and tumor necrosis, were used to attack the tumor vasculature, whereas the T-cell costimulator B7.1 (CD80), which costimulates T-cell proliferation via the CD28 pathway, was used to stimulate antitumor immunity. The injection of cDNA (60–180 µg) encoding B7.1 into large EL-4 tumors (0.8 cm in diameter) established in C57BL/6 mice, followed 24 h later by i.p. administration of either DMXAA (25 mg/kg) or FAA (300 mg/kg), resulted in complete tumor eradication within 2–6 weeks. In contrast, monotherapies were ineffective. Both vascular attack and B7.1 immunotherapy led to up-regulation of heat shock protein 70 on stressed and dying tumor cells, potentially augmenting immunotherapy. Remarkably, large tumors took on the appearance of a wound that rapidly ameliorated, leaving perfectly healed skin. Combined therapy was mediated by CD8+ T cells and natural killer cells, accompanied by heightened and prolonged antitumor cytolytic activity (P < 0.001), and by a marked increase in tumor cell apoptosis. Cured animals completely rejected a challenge of 1 x 107 parental EL-4 tumor cells but not a challenge of 1 x 104 Lewis lung carcinoma cells, demonstrating that antitumor immunity was tumor specific. Adoptive transfer of 2 x 108 splenocytes from treated mice into recipients bearing established (0.8 cm in diameter) tumors resulted in rapid and complete tumor rejection within 3 weeks. Although DMXAA and B7.1 monotherapies are complicated by a narrow range of effective doses, combined therapy was less dosage dependent. Thus, a broad range of amounts of B7.1 cDNA were effective in combination with 25 mg/kg DMXAA. In contrast, DMXAA, which has a very narrow range of high active doses, was effective at a low dose (18 mg/kg) when administered with a large amount (180 µg) of B7.1 cDNA. Importantly, combinational therapy generated heightened antitumor immunity, such that gene transfer of B7.1 into one tumor, followed by systemic DMXAA treatment, led to the complete rejection of multiple untreated tumor nodules established in the opposing flank. These findings have important implications for the future direction and utility of cancer immunotherapies aimed at harnessing patients’ immune responses to their own tumors.

The 6 by the power of 3 model for enhancing academic teachers' capacities for effective online teaching and learning: benefits, initiatives and future directions

To gain the full educational benefits of the major new investments in corporate technologies supporting online teaching and learning it is argued that a strategic, systems based approach of academic professional development (APD) is required. Such an approach requires a clear view of the key areas of potential and enduring teaching and learning benefit which can be realised from online developments, including an understanding of the changing role of the academic teacher in higher education, the identification of the desired professional capacities to educate online and the implementation of a number of coordinated initiatives to develop these professional capacities in order to engage constructively with the learning and technology opportunities. Based on previous work, we propose a '6' by the power of '3' model of Academic Professional Capacities Development for effective APD of online teaching and learning. The model can help inform the actions of policy makers, executives and practitioners in ways that promote an authentic learning organisation.

The TRIPS agreement and developing countries: a discussion of the paragraph 6 of the Doha declaration on public health

The Paragraph 6 solution arrived on 30th August 2003 to facilitate export of drugs to the countries which were not able to manufacture said drugs shows the total marginalization of developing countries in international treaty negotiations. A simple proposal by developing counties to use Article 30 of the TRIPS Agreement for such manufacture and export to non-manufacturing countries in order to avoid expensive litigations with the pharmaceutical multinationals took an ugly turn where not only the said proposal was totally rejected but export was added as one of the patenting rights in the TRIPS Agreement with payment of remuneration to patent holders. This introduction of export as one of the patenting rights was surrounded by a thicket of rules on the plea that such products would be diverted to ensure that the needing countries never acquire the requisite drugs. This article analyses the events leading to the establishment of the TRIPS Agreement, the elimination of developing countries from such negotiations through the use of suitably placed officials in the negotiating forums, the role of CEOs of the multinationals and the business NGOs such as International Intellectual Property Alliance and IPC (Intellectual Property Committee), epistemic community consisting of individuals such as Jacques Gorlin and Eric Smith and the subsequent development leading to the finalization of Para 6 Solution, which was an exact replication of events during the TRIPS negotiations. The analysis suggests that developing countries do not have any say in international negotiations and their agreements to such negotiations are essentially to legitimize their colonized existence.

What are grade 5 and 6 children buying from school canteens and what do parents and teachers think about it?

Objective: The foods sold in school canteens have a significant role to play in ensuring children consume a healthy balance of nutritious foods. However, no research to date has described the foods that Australian children are purchasing at school canteens, or the perceptions held about school canteens by students, teachers and parents.
Design: An exploratory cross-sectional survey was used to obtain information from students, parents and teachers through self completion questionnaires.
Subjects: There were 384 children aged nine to 12 years, 404 parents and 41 teachers involved.
Settings: The study was conducted in 12 primary schools in Victoria, Australia.
Main outcome measures: Types of foods purchased at school canteens, school canteen usage, parents' and teachers' perceptions of the role of the school and canteen in influencing children's eating habits.
Results: More than 50% of the children surveyed used the school canteen at least once per week. Children identified preference for unhealthy alternatives and availability as key barriers to choosing healthy foods at the canteen and suggested increased availability, advertisements and cost reductions as aids to purchasing healthy foods. Teachers placed more importance on the role of the canteen than parents did.
Statistical analyses: Frequencies, cross-tabulation analyses and chi square tests were undertaken using the SPSS 11 computer program.
Conclusions: These Victorian primary school children and their teachers, and to a lesser extent parents, had a clear understanding of healthy foods. Children and teachers identified barriers that prevent school canteens
from providing healthy foods. There is likely to be strong support from these stakeholders for novel health promoting policies.

Altering dietary nutrient intake that reduces glycogen content leads to phosphorylation of nuclear p38 MAP kinase in human skeletal muscle: association with IL-6 gene transcription during contraction

To determine the effect of glycogen availability and contraction on intracellular signaling and IL-6 gene transcription, eight males performed 60 min of exercise on two occasions: either with prior ingestion of a normal (Con) or low carbohydrate (LCHO) diet that reduced pre-exercise muscle glycogen content. Muscle biopsies were obtained and analyzed for IL-6 mRNA. In addition, nuclear proteins were isolated from the samples and analyzed for the mitogen- activated protein kinases (MAPK) c-jun amino-terminal kinase (JNK) 1 and 2 and p38 MAPK. Nuclear fractions were also analyzed for the phosphorylated forms of JNK (p-JNK) and p38 MAPK (p-p38 MAPK) and the abundance of the nuclear transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor kappa-β (NF-κβ). No differences were observed in the protein abundance of total JNK 1/2, p38 MAPK, NFAT, or NF-κβ before exercise, but the nuclear abundance of p-p38 MAPK was higher (P<0.05) in LCHO. Contraction resulted in an increase (P<0.05) in nuclear p-JNK 1/2, but there were no differences when comparing CON with LCHO. The fold increase in IL-6 mRNA with contraction was potentiated (P<0.05) in LCHO. A correlation between pre-exercise nuclear phosphorylated p38 MAPK and contraction-induced fold increase in IL-6 mRNA was performed, revealing a highly significant correlation (r=0.96; P<0.01). We next incubated L6 myotubes in ionomycin (a compound known to induce IL-6 mRNA) with or without the pyridinylimidazole p38 MAPK inhibitor SB203580. Treatments did not affect total nuclear p38 MAPK, but ionomycin increased (P<0.05) both nuclear p-p38 MAPK and IL-6 mRNA. The addition of SB203580 to ionomycin decreased (P<0.05) nuclear p-p38 MAPK and totally abolished (P<0.05) the ionomycin- induced increase in IL-6 mRNA. These data suggest that reduced carbohydrate intake that results in low intramuscular glycogen leads to phosphorylation of p38 MAPK at the nucleus. Furthermore, phosphorylation of p38 MAPK in the nucleus appears to be an upstream target for IL-6, providing new insights into the regulation of IL-6 gene transcription.

Examining physical activity among 5- to 6- and 10- to 12-year-old children: The children's leisure activities study

The aim of this study was to describe the type, frequency, duration, and intensity of children's physical activity and to examine differences by sex, age, and socioeconomic status. Participants consisted of 5- to 6-year-old (n = 291) and 10- to 12-year-old (n = 919) children and their parents who were taking part in the Children's Leisure Activities Study (CLASS). Parents completed proxy questionnaires about their child's activities, and all children wore an accelerometer for 8 days. Accelerometry data showed that younger children accumulated approximately 4 hrs of moderate- to vigorous-intensity physical activity (MVPA) per day, and older children accumulated approximately 2 hrs per day. Fewer than three-quarters of 10- to 12-year-old boys and less than half of 10- to 12-year-old girls recorded 120 min of MVPA per day. Significant differences in the number of activities, as well as the type and frequency of activities performed, were observed by age and sex. The findings indicate that physical activities that appeal to older girls, such as lifestyle-type noncompetitive activities, should be considered in the development of physical activity promotion strategies.

Omega 6 to omega 3 fatty acid imbalance early in life leads to persistant reductions in DHA levels in glycerophospholipids in rat hypothalamus even after long-term omega 3 fatty acid repletion

Failure to provide omega 3 fatty acids in the perinatal period results in alterations in nerve growth factor levels, dopamine production and  permanent elevations in blood pressure. The present study investigated whether changes in brain (i.e., hypothalamus) glycerophospholipid fatty acid profiles induced by a diet rich in omega 6 fatty acids and very low in alpha-linolenic acid (ALA) during pregnancy and the perinatal period could be reversed by subsequent feeding of a diet containing ALA. Female rats (6 per group) were mated and fed either a low ALA diet or a control diet containing ALA throughout pregnancy and until weaning of the pups at 3 weeks. At weaning, the pups (20 per group) remained on the diet of their mothers until 9 weeks, when half the pups were switched onto the other diet, thus generating four groups of animals. At 33 weeks, pups were killed, the hypothalamus dissected from the male rats and analysed for glycerophospholipid fatty acids. In the animals fed the diet with very little ALA and then re-fed the control diet containing high levels of ALA for 24 weeks, the DHA levels were still significantly less than the control values in PE, PS and PI fractions, by 9%, 18% and 34%, respectively. In this group, but not in the other dietary groups, ALA was detected in all glycerophospholipid classes at 0.2–1.7% of the total fatty acids. The results suggest that omega 6–3 PUFA imbalance early in life leads to irreversible changes in hypothalamic composition. The increased ALA and reduced DHA proportions in the animals re-fed ALA in later life are consistent with a dysfunction or down-regulation of the conversion of ALA to 18:4n-3 by the delta-6 desaturase.