Interactions of natural products with β-lactoglobulins, members of the lipocalin family

Natural products constitute an important source of new drugs. The bioavailability of the drugs depends on their absorption, distribution, metabolism and elimination. To achieve good bioavailability, the drug must be soluble in water, stable in the gastrointestinal tract and palatable. Binding proteins may improve the solubility of drug compounds, masking unwanted properties, such as bad taste, bitterness or toxicity, transporting or protecting these compounds during processing and storage. The focus of this thesis was to study the interactions, including ligand binding and the effect of pH and temperature, of bovine and reindeer β-lactoglobulin (βLG) with such compounds as retinoids, phenolic compounds as well as with compounds from plant extracts, and to investigate the transport properties of the βLG-ligand complex. To examine the binding interactions of different ligands to βLG, new methods were developed. The fluorescence binding method for the evaluation of ligand binding to βLG was miniaturized from a quartz cell to a 96-well plate. A method of ultrafiltration sampling combined with high-performance liquid chromatography was developed to assess the binding of compounds from extracts. The interactions of phenolic compounds or retinoids and βLG were investigated using the 96-well plate method. The majority of flavones, flavonols, flavanones and isoflavones and all of the retinoids included were shown to bind to bovine and reindeer βLG. Phenolic compounds, contrary to retinol, were not released at acidic pH. Those results suggest that βLG may have more binding sites, probably also on the surface of βLG. An extract from Camellia sinensis (L.) O. Kunze (black tea), Urtica dioica L. (nettle) and Piper nigrum (black pepper) were used to evaluate whether βLG could bind compounds from plant extracts. Piperine from P. nigrum was found to bind tightly and rutin from U. dioica weakly to βLG. No components from C. sinensis bound to βLG in our experiment. The uptake and membrane permeation of bovine and reindeer βLG, free and bound with retinol, palmitic acid and cholesterol, were investigated using Caco-2 cell monolayers. Both bovine and reindeer βLG were able to cross the Caco-2 cell membrane. Free and βLG-bound retinol and palmitic acid were transported equally, whereas cholesterol could not cross the Caco-2 cell monolayer free or bound to βLG. Our results showed that βLG can bind different natural product compounds, but cannot enhance transport of retinol, palmitic acid or cholesterol through Caco-2 cells. Despite this, βLG, as a water-soluble binding protein, may improve the solubility of natural compounds, possibly protecting them from early degradation and transporting some of them through the stomach. Furthermore, it may decrease their bad or bitter taste during oral administration of drugs or in food preparations. βLG can also enhance or decrease the health benefits of herbal teas and food preparations by binding compounds from extracts.

Protein turnover in malnourished patients with cystic fibrosis: Effects of elemental and nonelemental nutritional supplements

To evaluate the relative efficacy of nonele-mental versus semielemental enteral supplements for nutritional rehabilitation of cystic fibrosis (CF) patients, whole-body protein turnover using the [15N]glycine method was studied in nine malnourished CF patients during enteral feedings, in a block design study compar-ing a semielemental formula (Criticare), a higher protein density but nonelemental formula (Traumacal) (T), and a nonelemental formula that had been modified to become isocaloric and isonitrogenous to the semielemental formula (modified Traumacal, MT). No significant differences in rates of protein synthesis or catabolism were observed comparing the three formulas. However the higher protein density nonelemental formula resulted in higher net protein deposition compared to the other two formulas (T + 0.42 g kg-110 h-1versus 0.33 g kg-110 h-1for Criticare and-0.59 g kg-110 h-1for MT), although this was significant (p < 0.05) for the MT versus T comparison only. This study lends support to the use of less expensive nonelemental formulas for the nutritional management of malnourished patients with CF. © 1990 Raven Press Ltd, New York.

Intra and Inter-Molecular Communications Through Protein Structure Network

Communication within and across proteins is crucial for the biological functioning of proteins. Experiments such as mutational studies on proteins provide important information on the amino acids, which are crucial for their function. However, the protein structures are complex and it is unlikely that the entire responsibility of the function rests on only a few amino acids. A large fraction of the protein is expected to participate in its function at some level or other. Thus, it is relevant to consider the protein structures as a completely connected network and then deduce the properties, which are related to the global network features. In this direction, our laboratory has been engaged in representing the protein structure as a network of non-covalent connections and we have investigated a variety of problems in structural biology, such as the identification of functional and folding clusters, determinants of quaternary association and characterization of the network properties of protein structures. We have also addressed a few important issues related to protein dynamics, such as the process of oligomerization in multimers, mechanism on protein folding, and ligand induced communications (allosteric effect). In this review we highlight some of the investigations which we have carried out in the recent past. A review on protein structure graphs was presented earlier, in which the focus was on the graphs and graph spectral properties and their implementation in the study of protein structure graphs/networks (PSN). In this article, we briefly summarize the relevant parts of the methodology and the focus is on the advancement brought out in the understanding of protein structure-function relationships through structure networks. The investigations of structural/biological problems are divided into two parts, in which the first part deals with the analysis of PSNs based on static structures obtained from x-ray crystallography. The second part highlights the changes in the network, associated with biological functions, which are deduced from the network analysis on the structures obtained from molecular dynamics simulations.

Nucleotide Degradation Profiles of Iced Barramundi (Lates calcarifer) and Nile Perch (Lates niloticus) Muscle

Adenine nucleotides and their related compounds were determined in muscle extracts from two species of fish that were stored in ice after thawing. The fish were the closely related species, Australian barramundi (Lates calcarifer ) and Kenyan Nile perch (Lates niloticus ) which had different process histories. For all samples, adenine nucleotides did not exceed 6% of the total nucleotide pool. Inosine monophosphate (IMP) decreased steadily with storage. Hypoxanthine (Hx) was the major product of adenosine triphosphate (ATP) degradation in both barramundi and Nile perch, showing a steady increase with days of iced storage. The Hx level did not reach a maximum during the 9d storage period. The K-value also increased regularly with time of storage and for the later stages (i.e., 7 and 9d) and was significantly different (P < 0.01) for the two species. The iced storage life of these typical samples of barramundi and Nile perch was estimated to be 3d after thawing using a K-value of < 30% to indicate excellent quality. Despite the differences in process history the nucleotide profiles were remarkably similar during storage. This precludes the use of nucleotide levels as a means of differentiating between these species.

Laboratory investigation on the effects of overburden pressure, water, and time on slaking induced material property degradation of coal mine spoil

In open-cut strip mining, waste material is placed in-pit to minimise operational mine costs. Slope failures in these spoil piles pose a significant safety risk to personnel, along with a financial risk from loss of equipment and scheduling delays. It has been observed that most spoil pile failures occur when the pit has been previously filled with water and then subsequently dewatered. The failures are often initiated at the base of spoil piles where the material can undergo significant slaking (disintegration) over time due to overburden pressure and water saturation. It is important to understand how the mechanical properties of base spoil material are affected by slaking when designing safe spoil pile slope angles, heights, and dewatering rates. In this study, fresh spoil material collected from a coal mine in Brown Basin Coalfield of Queensland, Australia was subjected to high overburden pressure (0 – 900 kPa) under saturated condition and maintained over a period of time (0 – 6 months) allowing the material to slake. To create the above conditions, laboratory designed pressure chambers were used. Once a spoil sample was slaked under certain overburden pressure over a period of time, it was tested for classification, permeability, and strength properties. Results of this testing program suggested that the slaking of saturated coal mine spoil increase with overburden pressure and the time duration over which the overburden pressure was maintained. Further, it was observed that shear strength and permeability of spoil decreased with increase in spoil slaking.

Thermochemical and kinetic studies on the effects of cobalt salicylate on the oxidative degradation of polystyrene

The effect of cobalt salicylate on the oxidative degradation and ignition of polystyrene has been studied. It was found that cobalt salicylate sensitizes both the degradation and ignition of polystyrene by facilitating electron-transfer processes in the propagation step. From thermochemical and kinetic studies it was found that the cobalt ion, owing to its ability to exist in variable valence states, promotes electron transfer in the propagation step of polymer degradation, increasing the rate of propagation and consequently the overall rate. Using solid-phase thermal ignition theory, an attempt has been made to explain the sensitization of ignition by the cobalt ion.

Perylene-based profluorescent nitroxides for the rapid monitoring of polyester degradation upon weathering: An assessment

A profluorescent nitroxide possessing an isoindoline nitroxide moiety linked to a perylene fluorophore was developed to monitor radical mediated degradation of melamine-formaldehyde crosslinked polyester coil coatings in an industry standard accelerated weathering tester. Trapping of polyester-derived radicals (most likely C-radicals) that are generated during polymer degradation leads to fluorescent closed-shell alkoxy amines, which was used to obtain time-dependent degradation profiles to assess the relative stability of different polyesters towards weathering. The nitroxide probe couples excellent thermal stability and satisfactory photostability with high sensitivity and enables detection of free radical damage in polyesters under conditions that mimic exposure to the environment on a time scale of hours rather than months or years required by other testing methods. There are indications that the profluorescent nitroxide undergoes partial photo-degradation in the absence of polymer-derived radicals. Unexpectedly, it was also found that UV-induced fragmentation of the NO–C bond in closed-shell alkoxy amines leads to regeneration of the profluorescent nitroxide and the respective C-radical. The maximum fluorescence intensity that could be achieved with a given probe concentration is therefore not only determined by the amount of polyester radicals formed during accelerated weathering, but also by the light-driven side reactions of the profluorescent nitroxide and the corresponding alkoxy amine radical trapping products. Studies to determine the optimum probe concentration in the polymer matrix revealed that aggregation and re-absorption effects lowered the fluorescence intensity at higher concentrations of the profluorescent nitroxide, but too low probe concentrations, where these effects would be avoided, were not sufficient to trap the amount of polyester radicals formed upon weathering. The optimized experimental conditions were used to assess the impact of temperature and UV irradiance on polymer degradation during accelerated weathering.

Vaccination with an Insulin-like Growth Factor Binding Protein-1 (IGFBP-1)-bBased Vaccine Improves Growth in Feed-Restricted Brahman Steers

Dry-season weight loss in grazing cattle in northern Australia has been attenuated using a number of strategies (Hunter and Vercoe, 1987, Sillence et al. 1993, Gazzola and Hunter, 1999). Furthermore, the potential to improve efficiency of feed utilisation (and thus, dry-season performance) in ruminants through conventional modulation of the insulin-like growth factor (IGF) axis (Oddy and Owens, 1997, Hill et al., 1999) and through immunomodulation of the IGF axis (Hill et al., 1998a,b) has been demonstrated. The present study investigated the use of a vaccine directed against IGFBP-1 in Brahman steers which underwent a period of nutritional restriction followed by a return to wet-season grazing.

Effect of pregnant mare serum gonadotropin on the induction and degradation of FSH and LH receptors in the granulosa cell of the immature rat

The relative induction of FSH and LH receptors in the granulosa cells of immature rat ovary by pregnant mare serum gonadotropin (PMSG) has been studied. A single injection of PMSG (15 IU) brought about a 3- and 12-fold increase in FSH and LH receptor concentration,respectively, in the granulosa cells. Maximal concentration was reached by 72 h but the receptor levels showed a sharp decline during the next 24–48 h. The kinetic properties of the newly formed FSH receptors were indistinguishable from the pre-existing ones. The induced FSH receptors were functional as demonstrated by an increase in the in vitro responsiveness of the cells to exogenous FSH in terms of progesterone production. Treatment of immature rats with cyanoketone, an inhibitor of Δ5,3β-hydroxysteroid dehydrogenase, prior to PMSG injection effectively reduced the PMSG-stimulated increase in the serum estradiol, uterine weight and LH receptors but had no effect on the FSH receptor induction. The ability of PMSG to induce gonadotropin receptors can be arrested at any given time by injecting its antibody, thereby suggesting a continuous need for the hormonal inducer. Estrogen in the absence of the primary inducer was unable to maintain the induced LH and FSH receptor concentration. Inhibition of prostaglandin synthesis using indomethacin also had no effect on either the induction or degradation of gonadotropin receptors. Administration of PMSG antiserum, 48 h after PMSG injection, brought about a rapid decline in the induced receptors over the next 24 h, with a rate constant and \iota 1/2 of 0.078 h−1 and 8.9 h for FSH receptors and 0.086 h−1 and 8.0 h for the LH receptors, respectively.

Short-term nutritional supplementation during management of pulmonary exacerbations in cystic fibosis: A controlled study, including effects of protein turnover

Effects of nutritional supplements on minimizing weight loss and abnormalities of protein turnover during pulmonary exacerbations in cystic fibrosis (CF) were studied by controlled trial. Patients received pulmonary therapy and either standard diet (n = 10) or adjunctive enteral supplements (n = 12). Initial protein turnover, measured by [15N]glycine kinetics, showed alterations of protein synthesis (P Syn) and catabolism (P Cat), which correlated with the degree of underweight, and negligible net protein deposition (P Dep). With treatment both groups had significant increases in mean body weight and forced expiratory volume in 1 s, expressed as percent predicted value for height (FEV1) by 3 wk, but a significant correlation between initial underweight and subsequent weight gain was observed only in supplemented patients. Mean P Syn and P Dep increased significantly (p < 0.001) only in the supplemented group. Pulmonary exacerbations in CF have important adverse effects on body-protein metabolism, similar to changes in protein-energy malnutrition and infection. These effects are reversed by short-term nutritional support. Strategic nutritional intervention should thus be considered in management, especially in malnourished patients.

Sustaining productivity of a Vertosol at Warra, Queensland, with fertilisers, no-tillage or legumes. 8. Effect of duration of lucerne ley on soil nitrogen and water, wheat yield and protein.

Soil nitrogen (N) supply in the Vertosols of southern Queensland, Australia has steadily declined as a result of long-term cereal cropping without N fertiliser application or rotations with legumes. Nitrogen-fixing legumes such as lucerne may enhance soil N supply and therefore could be used in lucerne-wheat rotations. However, lucerne leys in this subtropical environment can create a soil moisture deficit, which may persist for a number of seasons. Therefore, we evaluated the effect of varying the duration of a lucerne ley (for up to 4 years) on soil N increase, N supply to wheat, soil water changes, wheat yields and wheat protein on a fertility-depleted Vertosol in a field experiment between 1989 and 1996 at Warra (26degrees 47'S, 150degrees53'E), southern Queensland. The experiment consisted of a wheat-wheat rotation, and 8 treatments of lucerne leys starting in 1989 (phase 1) or 1990 (phase 2) for 1,2,3 or 4 years duration, followed by wheat cropping. Lucerne DM yield and N yield increased with increasing duration of lucerne leys. Soil N increased over time following 2 years of lucerne but there was no further significant increase after 3 or 4 years of lucerne ley. Soil nitrate concentrations increased significantly with all lucerne leys and moved progressively downward in the soil profile from 1992 to 1995. Soil water, especially at 0.9-1.2 m depth, remained significantly lower for the next 3 years after the termination of the 4 year lucerne ley than under continuous wheat. No significant increase in wheat yields was observed from 1992 to 1995, irrespective of the lucerne ley. However, wheat grain protein concentrations were significantly higher under lucerne-wheat than under wheat wheat rotations for 3-5 years. The lucerne yield and soil water and nitrate-N concentrations were satisfactorily simulated with the APSIM model. Although significant N accretion occurred in the soil following lucerne leys, in drier seasons, recharge of the drier soil profile following long duration lucerne occurred after 3 years. Consequently, 3- and 4-year lucerne-wheat rotations resulted in more variable wheat yields than wheat-wheat rotations in this region. The remaining challenge in using lucerne-wheat rotations is balancing the N accretion benefits with plant-available water deficits, which are most likely to occur in the highly variable rainfall conditions of this region.

Inverse Correlation between Vitamin D and C-Reactive Protein in Newborns

Some studies suggested that adequate vitamin D might reduce inflammation in adults. However, little is known about this association in early life. We aimed to determine the relationship between cord blood 25-hydroxyvitamin D (25(OH)D) and C-reactive protein (CRP) in neonates. Cord blood levels of 25(OH)D and CRP were measured in 1491 neonates in Hefei, China. Potential confounders including maternal sociodemographic characteristics, perinatal health status, lifestyle, and birth outcomes were prospectively collected. The average values of cord blood 25(OH)D and CRP were 39.43 nmol/L (SD = 20.35) and 6.71 mg/L (SD = 3.07), respectively. Stratified by 25(OH)D levels, per 10 nmol/L increase in 25(OH)D, CRP decreased by 1.42 mg/L (95% CI: 0.90, 1.95) among neonates with 25(OH)D <25.0 nmol/L, and decreased by 0.49 mg/L (95% CI: 0.17, 0.80) among neonates with 25(OH)D between 25.0 nmol/L and 49.9 nmol/L, after adjusting for potential confounders. However, no significant association between 25(OH)D and CRP was observed among neonates with 25(OH)D ≥50 nmol/L. Cord blood 25(OH)D and CRP levels showed a significant seasonal trend with lower 25(OH)D and higher CRP during winter-spring than summer-autumn. Stratified by season, a significant linear association of 25(OH)D with CRP was observed in neonates born in winter-spring (adjusted β = −0.11, 95% CI: −0.13, −0.10), but not summer-autumn. Among neonates born in winter-spring, neonates with 25(OH)D <25 nmol/L had higher risk of CRP ≥10 mg/L (adjusted OR = 3.06, 95% CI: 2.00, 4.69), compared to neonates with 25(OH)D ≥25 nmol/L. Neonates with vitamin D deficiency had higher risk of exposure to elevated inflammation at birth.