Current concepts in membrane protein reconstitution

The reconstitution of integral proteins into artificial lipid vesicles is largely prompted by the complexity of most biological membranes and the inherent difficulty of studying individual components in situ. Ideally, therefore, the reconstituted system should consist of a single protein in a lipid matrix which mimics the native membrane in all but its diversity. While such an approach allows individual components of a complex system to be studied in isolation it should also be sufficiently versatile to permit the generation of increasingly sophisticated multicomponent models. From the considerable number of reconstitution techniques which have been developed I have tried in this review to identify those characteristics of a particular system which maximise both the information it can provide and its versatility.     

An assessment of the impact of Raman based glucose feedback control on CHO cell bioreactor process development

Process analytical technology (PAT) tools such as Raman Spectroscopy have become established tools for real time measurement of CHO cell bioreactor process variables and are aligned with the QbD approach to manufacturing. These tools can have a significant impact on process development if adopted early, creating an end-to-end PAT/QbD focused process. This study assessed the impact of Raman based feedback control on early and late phase development bioreactors by using a Raman based PLS model and PAT management system to control glucose in two CHO cell line bioreactor processes. The impact was then compared to bioreactor processes which used manual bolus fed methods for glucose feed delivery. Process improvements were observed in terms of overall bioreactor health, product output and product quality. Raman controlled batches for Cell Line 1 showed a reduction in glycation of 43.4% and 57.9%, respectively. Cell Line 2 batches with Raman based feedback control showed an improved growth profile with higher VCD and viability and a resulting 25% increase in overall product titer with an improved glycation profile. The results presented here demonstrate that Raman spectroscopy can be used in both early and late-stage process development and design for consistent and controlled glucose feed delivery.     

Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New Step in Neutrophil Recruitment

Inflammation is a physiological response to tissue trauma or infection, but leukocytes, which are the effector cells of the inflammatory process, have powerful tissue remodelling capabilities. Thus, to ensure their precise localisation, passage of leukocytes from the blood into inflamed tissue is tightly regulated. Recruitment of blood borne neutrophils to the tissue stroma occurs during early inflammation. In this process, peptide agonists of the chemokine family are assumed to provide a chemotactic stimulus capable of supporting the migration of neutrophils across vascular endothelial cells, through the basement membrane of the vessel wall, and out into the tissue stroma. Here, we show that, although an initial chemokine stimulus is essential for the recruitment of flowing neutrophils by endothelial cells stimulated with the inflammatory cytokine tumour necrosis factor-α, transit of the endothelial monolayer is regulated by an additional and downstream stimulus. This signal is supplied by the metabolism of the omega-6-polyunsaturated fatty acid (n-6-PUFA), arachidonic acid, into the eicosanoid prostaglandin-D₂ (PGD₂) by cyclooxygenase (COX) enzymes. This new step in the neutrophil recruitment process was revealed when the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an alternative substrate for COX enzymes, leading to the generation of PGD₃. This alternative series eicosanoid inhibited the migration of neutrophils across endothelial cells by antagonising the PGD₂ receptor. Here, we describe a new step in the neutrophil recruitment process that relies upon a lipid-mediated signal to regulate the migration of neutrophils across endothelial cells. PGD₂ signalling is subordinate to the chemokine-mediated activation of neutrophils, but without the sequential delivery of this signal, neutrophils fail to penetrate the endothelial cell monolayer. Importantly, the ability of the dietary n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected level of regulation in the migration of inflammatory leukocytes, it also contributes to our understanding of the interactions of this bioactive lipid with the inflammatory system. Moreover, it indicates the potential for novel therapeutics that target the inflammatory system with greater affinity and/or specificity than supplementing the diet with n-3-PUFAs.     

Patterns of association at feeder stations for Common Pheasants released into the wild: sexual segregation by space and time

Sexual segregation is common and can occur when sexes occupy different habitats, and/or when sexes aggregate assortatively within the same habitats. However, it is rarely studied in birds, with most previous work concentrating on differential settlement by the sexes in discrete habitats, often separated by large distances. Little attention has been paid to patterns of segregation within the same site. We reared 200 Common Pheasants Phasianus colchicus and released them onto a relatively small site of 250 ha and recorded their patterns of association and differential use of artificial feeders in space and time. Particular feeders were preferred by one sex, although we found no features of the local habitat which explained such preferences. Furthermore, we found sex differences in the use of feeders throughout the day, with females preferentially visiting them in the morning and the proportion of females visiting feeders increasing as the year progressed. Social network analyses found that in the first month after release into the wild, females did not associate strongly with other females, which was surprising as, prior to release, females have been shown to associate with other females in both semi‐natural conditions and when tested in isolation. However, sexual segregation was clearly seen after 1 month of being released and became more pronounced as the year progressed. Females associated with other females from November to February, whereas males avoided other males over this same period. Sexes became less likely to associate with one another in 5 of the 6 months monitored. Such avoidance observed in males suggests that they start to form territories much sooner than previously thought. Pheasants exhibit clear patterns of fine‐scale sexual segregation based on space and time, which was observed in their social preferences at feeding sites. Such detailed fine‐scale segregation is rarely observed in birds.     

Bacteria and yeasts associated with Sirex noctilio

Five genera of bacteria and yeasts commonly occur in the oviposition drills and larval galleries of Sirex noctilio. In exceptional circumstances, some of these microorganisms may be pathogenic while the yeast, Saccharomyces sp., is involved in the production of attractants of the rhyssine parasitoids Rhyssa and Megarhyssa spp. A possible role in larval nutrition is suggested but these microorganisms have no role in the phytotoxic effects of Sirex attack.     

Dorsomedial hypothalamic and brainstem pathways controlling thermogenesis in brown adipose tissue

1. The rostral medullary raphe pallidus contains sympathetic premotor neurons controlling thermogenesis in brown adipose tissue (BAT).

2. Disinhibition of neurons in the dorsomedial hypothalamus (DMH) stimulates BAT thermogenesis through activation of neurons in raphe pallidus.

3. An increase in BAT sympathetic outflow and BAT thermogenesis following microinjection of prostaglandin E2 into the preoptic area requires activation of both DMH neurons and raphe pallidus neurons.

4. DMH contains a population of neurons receiving a tonically- active GABAergic inhibition which mediate increases in BAT thermogenesis through stimulation of BAT sympathetic premotor neurons in raphe pallidus.     

ABRF ESRG 2005 study: identification of seven modified amino acids by Edman sequencing.

Identification of modified amino acids can be a challenging part for Edman degradation sequence analysis, largely because they are not included among the commonly used phenylthiohydantion amino acid standards. Yet many can have unique retention times and can be assigned by an experienced researcher or through the use of a guide showing their typical chromatography characteristics. The Edman Sequencing Research Group (ESRG) 2005 study is a continuation of the 2004 study, in which the participating laboratories were provided a synthetic peptide and asked to identify the modified amino acids present in the sequence. The study sample provided an opportunity to sequence a peptide containing a variety of modified amino acids and note their retention times relative to the common amino acids. It also allowed the ESRG to compile the chromatographic properties and intensities from multiple instruments and tabulate an average elution position for these modified amino acids on commonly used instruments. Participating laboratories were given 2000 pmoles of a synthetic peptide, 18 amino acids long, containing the following modified amino acids: dimethyl- and trimethyl-lysine, 3-methyl-histidine, N-carbamyl-lysine, cystine, N-methyl-alanine, and isoaspartic acid. The modified amino acids were interspersed with standard amino acids to help in the assessment of initial and repetitive yields. In addition to filling in an assignment sheet, which included retention times and peak areas, participants were asked to provide specific details about the parameters used for the sequencing run. References for some of the modified amino acid elution characteristics were provided and the participants had the option of viewing a list of the modified amino acids present in the peptide at the ESRG Web site. The ABRF ESRG 2005 sample is the seventeenth in a series of studies designed to aid laboratories in evaluating their abilities to obtain and interpret amino acid sequence data.     

Trichinella spiralis: major histocompatibility complex-associated elimination of encysted muscle larvae in swine

A heretofore undescribed host-mediated reactivity against encapsulated muscle larvae (ML) of the nematode Trichinella spiralis is reported. Inbred miniature swine (NIH minipigs) of three independent SLA phenotypes, which received a primary oral dose of 300 T. spiralis ML, successfully resisted a secondary infection of 10,000 ML; however, only pigs of the SLAa/a phenotype exhibited an unusual and highly significant reduction in the numbers of encysted ML from the primary infection (P less than 0.0003). This initial anti-encysted ML reactivity was confirmed in subsequent trials by comparing the prechallenge ML burdens with the reduced ML numbers in primary-infected aa pigs after challenge. Analyses of inbred strains of mice, selected for major histocompatibility type and for resistance or susceptibility to infection with T. spiralis, showed no such anti-encysted ML response. Because elimination of encysted T. spiralis ML had been accomplished previously only through selected drug regimens, our demonstration of a nonpharmacological, host-mediated reactivity against this stage of the parasite in swine highlights the importance of MHC genes in regulating disease resistance in a livestock species.