Stale flavour volatiles in Australian commercial UHT milk during storage

Methyl ketones, aldehydes and free saturated fatty acids were measured in the headspace of samples of two indirectly processed and two directly processed Australian commercial UHT milks during room temperature storage for 16 weeks. The analytes were isolated using headspace solid phase microextraction and analysed by gas chromatography coupled with flame ionisation detection. All methyl ketones and aldehydes increased during storage, With free saturated fatty acids exhibiting little change. On average, the total methyl ketone and aldehyde concentrations in the indirectly processed UHT milks were higher than those in the directly processed samples. A strong correlation was found between the concentration of methyl ketones and various heat indices (furosine, lactulose and undenatured whey proteins) in the milk samples.

Novel pMDI formulations for pulmonary delivery of proteins

In this paper, we demonstrate that co-spray-drying a model protein with sodium carboxymethylcellulose (NaCMC) protects protein integrity during spray-drying, and that the resultant spray-dried powders can be successfully dispersed in hydrofluoroalkane (HFA) propellant to prepare pressurised metered dose (pMDI) formulations that exhibit high respirable fractions. The spray-dried powders were formulated as HFA-134a pMDI suspensions in the absence of any other excipients (e.g. surfactants) or co-solvents (e.g. ethanol). The in vitro aerosolisation profile of these systems was assessed using the twin stage impinger; fine particle fractions (FPF) ≥50% of the recovered dose were obtained. Following storage for five months, the aerosolisation performance was reassessed; the NaCMC-free formulation demonstrated a significant decrease in FPF, whereas the performance of the NaCMC-modified formulations was statistically equivalent to their initial performance. Thus, formulation of pMDI suspensions using NaCMC-based spray-dried powders is a promising approach for the pulmonary delivery of proteins and peptides. © 2009 Elsevier B.V. All rights reserved.

The use of sodium carboxymethylcellulose in the preparation of spray-dried proteins for pulmonary drug delivery

The use of sodium carboxymethylcellulose (NaCMC) as a spray-drying excipient in the preparation of inhalable formulations of proteins was investigated, using alkaline phosphatase as a model functional protein. Two spray-dried powders were investigated: a control powder comprising 100% (w/w) alkaline phosphatase and a test powder comprising 67% (w/w) NaCMC and 33% (w/w) alkaline phosphatase. Following physicochemical characterisation, the powders were prepared as both dry powder inhaler (DPI) and pressurised metered dose inhaler (pMDI) formulations. The aerosolisation performance of the formulations was assessed using a Multi-Stage Liquid Impinger, both immediately after preparation and over a 16-week storage period. Formulating the control powder as a DPI resulted in a poor fine particle fraction (FPF: 10%), whereas the FPF of the NaCMC-modified DPI formulation was significantly greater (47%). When the powders were formulated as pMDI systems, the control and NaCMC-modified powders demonstrated FPFs of 52% and 55%, respectively. Following storage, reduced FPF was observed for all formulations except the NaCMC-modified pMDI system; the performance of this formulation following storage was statistically equivalent to that immediately following preparation. Co-spray-drying proteins and peptides with NaCMC may therefore offer an alternative method for the preparation of stable and respirable pMDI formulations for pulmonary delivery. © 2010 Elsevier B.V.

Exploring several nonconventional interactions of ice-binding proteins

Traditionally, ice-binding proteins (IBPs), also known as antifreeze proteins (AFPs), have been defined by two universal activities: ice recrystallization inhibition and thermal hysteresis. However, there remains the possibility IBPs have other complementary functions given the diversity found within this protein group. This thesis explores some of these in both natural and applied settings, in the hopes of furthering our understanding of this remarkable group of proteins. Plant IBPs could function as part of a defensive strategy against ice nucleators produced by certain pathogens. To assess this hypothesis, recombinant IBPs from perennial ryegrass and purple false brome were combined with the ice nucleation protein (INP) from the plant pathogen, Pseudomonas syringae. Strikingly, the plant proteins depressed the freezing point of the bacterial INP, while a fish AFP could not, nor did the INPs have any effect on IBP activity. Thus, the interaction between these two different proteins suggests a role in plant defensive strategies against pathogenic bacteria as another IBP function. In addition, the potential use of hyperactive insect IBPs in organ preservation was investigated. Current kidney preservation techniques involve storing the organ at 4 °C for a maximum of 24 h prior to transplantation. Extending this “safe” time would have profound effects on renal transplants, however, ischemic injury is prevalent when storage periods are prolonged. Experiments described here allowed subzero preservation for 72 h with the addition of a beetle IBP to CryoStasis® solution. Kidneys stored using the traditional technique for 24 h and the method developed here for 72 h showed similar levels of biomarker enzymes, underscoring the potential utility of insect IBPs for future transplant purposes. Finally, IBP function in the freeze-tolerant gall fly, Eurosta solidaginis, was examined. Larvae representing the mid-autumn stage displayed ice-binding activity, suggesting an IBP is being expressed, possibly as a protective measure against freezing damage when fall temperatures can unpredictably drop. IBP activity was also observed in the larvae’s host plant, Solidago spp. Mass spectrometry analysis of ice-affinity purified plant extracts provided three candidate pathogenesis-related proteins that could be responsible for the detected activity, further demonstrating additional functions of IBPs.

Biochemical studies of postsynaptic density signaling proteins with a focus on synaptic GTPase activating protein and PDZ domains

Memory storage in the brain involves adjustment of the strength of existing synapses and formation of new neural networks. A key process underlying memory formation is synaptic plasticity, the ability of excitatory synapses to strengthen or weaken their connections in response to patterns of activity between their connected neurons. Synaptic plasticity is governed by the precise pattern of Ca²⁺ influx through postsynaptic N-methyl-D-aspartate-type glutamate receptors (NMDARs), which can lead to the activation of the small GTPases Ras and Rap. Differential activation of Ras and Rap acts to modulate synaptic strength by promoting the insertion or removal of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptors (AMPARs) from the synapse. Synaptic GTPase activating protein (synGAP) regulates AMPAR levels by catalyzing the inactivation of GTP-bound (active) Ras or Rap. synGAP is positioned in close proximity to the cytoplasmic tail regions of the NMDAR through its association with the PDZ domains of PSD-95. SynGAP’s activity is regulated by the prominent postsynaptic protein kinase, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5), a known binding partner of CaMKII. Modulation of synGAP’s activity by phosphorylation may alter the ratio of active Ras to Rap in spines, thus pushing the spine towards the insertion or removal of AMPARs, subsequently strengthening or weakening the synapse. To date, all biochemical studies of the regulation of synGAP activity by protein kinases have utilized impure preparations of membrane bound synGAP. Here we have clarified the effects of phosphorylation of synGAP on its Ras and Rap GAP activities by preparing and utilizing purified, soluble recombinant synGAP, Ras, Rap, CaMKII, CDK5, PLK2, and CaM. Using mass spectrometry, we have confirmed the presence of previously identified CaMKII and CDK5 sites in synGAP, and have identified novel sites of phosphorylation by CaMKII, CDK5, and PLK2. We have shown that the net effect of phosphorylation of synGAP by CaMKII, CDK5, and PLK2 is an increase in its GAP activity toward HRas and Rap1. In contrast, there is no effect on its GAP activity toward Rap2. Additionally, by assaying the GAP activity of phosphomimetic synGAP mutants, we have been able to hypothesize the effects of CDK5 phosphorylation at specific sites in synGAP. In the course of this work, we also found, unexpectedly, that synGAP is itself a Ca²⁺/CaM binding protein. While Ca²⁺/CaM binding does not directly affect synGAP activity, it causes a conformational change in synGAP that increases the rate of its phosphorylation and exposes additional phosphorylation sites that are inaccessible in the absence of Ca²⁺/CaM.

The postsynaptic density (PSD) is an electron-dense region in excitatory postsynaptic neurons that contains a high concentration of glutamate receptors, cytoskeletal proteins, and associated signaling enzymes. Within the PSD, three major classes of scaffolding molecules function to organize signaling enzymes and glutamate receptors. PDZ domains present in the Shank and PSD-95 scaffolds families serve to physically link AMPARs and NMDARs to signaling molecules in the PSD. Because of the specificity and high affinity of PDZ domains for their ligands, I reasoned that these interacting pairs could provide the core components of an affinity chromatography system, including affinity resins, affinity tags, and elution agents. I show that affinity columns containing the PDZ domains of PSD-95 can be used to purify active PDZ domain-binding proteins to very high purity in a single step. Five heterologously expressed neuronal proteins containing endogenous PDZ domain ligands (NMDAR GluN2B subunit Tail, synGAP, neuronal nitric oxide synthase PDZ domain, cysteine rich interactor of PDZ three and cypin) were purified using PDZ domain resin, with synthetic peptides having the sequences of cognate PDZ domain ligands used as elution agents. I also show that conjugation of PDZ domain-related affinity tags to Proteins Of Interest (POIs) that do not contain endogenous PDZ domains or ligands does not alter protein activity and enables purification of the POIs on PDZ domain-related affinity resins.

Natural variation in expression of genes associated with carotenoid biosynthesis and accumulation in cassava (Manihot esculenta Crantz) storage root.

ABSTRACT: BACKGROUND: Cassava (Manihot esculenta Crantz) storage root provides a staple food source for millions of people worldwide. Increasing the carotenoid content in storage root of cassava could provide improved nutritional and health benefits. Because carotenoid accumulation has been associated with storage root color, this study characterized carotenoid profiles, and abundance of key transcripts associated with carotenoid biosynthesis, from 23 landraces of cassava storage root ranging in color from white-to-yellow-to-pink. This study provides important information to plant breeding programs aimed at improving cassava storage root nutritional quality. RESULTS: Among the 23 landraces, five carotenoid types were detected in storage root with white color, while carotenoid types ranged from 1 to 21 in storage root with pink and yellow color. The majority of storage root in these landraces ranged in color from pale-to-intense yellow. In this color group, total ß-carotene, containing all-E-, 9-Z-, and 13-Z-ß-carotene isomers, was the major carotenoid type detected, varying from 26.13 to 76.72 %. Although no ?-carotene was observed, variable amounts of a ?-ring derived xanthophyll, lutein, was detected; with greater accumulation of ?-ring xanthophylls than of ß-ring xanthophyll. Lycopene was detected in a landrace (Cas51) with pink color storage root, but it was not detected in storage root with yellow color. Based on microarray and qRT-PCR analyses, abundance of transcripts coding for enzymes involved in carotenoid biosynthesis were consistent with carotenoid composition determined by contrasting HPLC-Diode Array profiles from storage root of landraces IAC12, Cas64, and Cas51. Abundance of transcripts encoding for proteins regulating plastid division were also consistent with the observed differences in total ß-carotene accumulation. CONCLUSIONS: Among the 23 cassava landraces with varying storage root color and diverse carotenoid types and profiles, landrace Cas51 (pink color storage root) had low LYCb transcript abundance, whereas landrace Cas64 (intense yellow storage root) had decreased HYb transcript abundance. These results may explain the increased amounts of lycopene and total ß-carotene observed in landraces Cas51 and Cas64, respectively. Overall, total carotenoid content in cassava storage root of color class representatives were associated with spatial patterns of secondary growth, color, and abundance of transcripts linked to plastid division. Finally, a partial carotenoid biosynthesis pathway is proposed.

Changes in enzymatic activity, accumulation of proteins and softening of persimmon (Diospyros kaki Thunb.) flesh as a function of pre-cooling acclimatization.

One of the major causes of ?Fuyu? persimmon loss after cold storage (CS) is the breakdown of its flesh, which results in the production of a translucent fruit (a water-soaked fruit). It is believed that the cause of this disturbance is linked to disorganization of the cytoskelet and endomembrane system, which changes the synthesis and transport of proteins and metabolites, resulting in incomplete ripening. To test this hypothesis, ?Fuyu? persimmon was subjected to three different postharvest treatments (T): Control ? harvested and kept at 23±3 ◦C and relative humidity (RH) of 85±5% (room temperature, RT) for 12 days, T1 ? harvested and kept under cold storage (CS) (1±1 ◦C and RH of 85±5%) for 30 days followed by RT storage for 2 days, T2 ? kept under RT for 2 days (acclimatization) followed by CS for 30 days. Control and T2 resulted in fruit with decreased flesh firmness (FF), and increased soluble solids (SS) and ascorbic acid (AA) contents. In these fruit the activity of endo-1,4-ß-glucanase (endo-1,4-ß-gluc), pectin methylesterase (PME), polygalacturonase (PG) and ß-galactosidase (ß-gal) increased. T1 resulted in translucent fruit with decreased FF, without any enzymatic activity changes, probably due to the physical disruption of the cytoskeleton. Further, there was an increased content of proteins corresponding to expansins in fruit kept under Control and T2 conditions, which suggests that these conditions do contribute to the synthesis and/or transport of proteins involved in the process of solubilization of the cell wall. In these fruit, there was also a major accumulation of gene transcripts corresponding to heat shock proteins (HSPs) of organelles related to endomembrane, which suggests participation of these genes in the prevention of damage caused by cold conditions. These data proved the hypotheses that acclimatization contributes to the expression of HSPs, and synthesis and transportat of proteins involved in the solubilization of the cell wall. The expression of these genes results in the normal ripening of the persimmon, as confirmed by the evolution of ethylene production.

Public acceptance of carbon dioxide capture and storage in a proposed demonstration area

With the rising levels of CO2 in the atmosphere, low-emission technologies with carbon dioxide capture and storage (CCS) provide one option for transforming the global energy infrastructure into a more environmentally, climate sustainable system. However, like many technology innovations, there is a social risk to the acceptance of CCS. This article presents the findings of an engagement process using facilitated workshops conducted in two communities in rural Queensland, Australia, where a demonstration project for IGCC with CCS has been announced. The findings demonstrate that workshop participants were concerned about climate change and wanted leadership from government and industry to address the issue. After the workshops, participants reported increased knowledge and more positive attitudes towards CCS, expressing support for the demonstration project to continue in their local area. The process developed is one that could be utilized around the world to successfully engage communities on the low carbon emission technology options.

Ceramic membranes for separation of proteins and DNA by in situ growth of alumina nanofibres with a nanomesh of metal oxide nanofibres

Ceramic membranes were fabricated by in situ synthesis of alumina nanofibres in the pores of an alumina support as a separation layer, and exhibited a high permeation selectivity for bovine serum albumin relative to bovine hemoglobin (over 60 times) and can effectively retain DNA molecules at high fluxes.

A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins

A surface plasmon resonance-based solution affinity assay is described for measuring the Kd of binding of heparin/heparan sulfate-binding proteins with a variety of ligands. The assay involves the passage of a pre-equilibrated solution of protein and ligand over a sensor chip onto which heparin has been immobilised. Heparin sensor chips prepared by four different methods, including biotin–streptavidin affinity capture and direct covalent attachment to the chip surface, were successfully used in the assay and gave similar Kd values. The assay is applicable to a wide variety of heparin/HS-binding proteins of diverse structure and function (e.g., FGF-1, FGF-2, VEGF, IL-8, MCP-2, ATIII, PF4) and to ligands of varying molecular weight and degree of sulfation (e.g., heparin, PI-88, sucrose octasulfate, naphthalene trisulfonate) and is thus well suited for the rapid screening of ligands in drug discovery applications.