Divergent outcomes following transcytosis of IgG targeting intracellular and extracellular chlamydial antigens

Antibodies can play a protective but non-essential role in natural chlamydial infections dependent on antigen specificity and antibody isotype. IgG is the dominant antibody in both male and female reproductive tract mucosal secretions, and is bi-directionally trafficked across epithelia by the neonatal Fc receptor (FcRn). Using physiologically relevant pH-polarized epididymal epithelia grown on Transwells®, IgG specifically targeting an extracellular chlamydial antigen; the Major Outer Membrane Protein (MOMP), enhanced uptake and translocation of infection at pH 6-6.5 but not at neutral pH. This was dependent on FcRn expression. Conversely, FcRn-mediated transport of IgG targeting the intracellular chlamydial inclusion membrane protein A (IncA), induced aberrant inclusion morphology, recruited autophagic proteins independent of lysosomes, and significantly reduced infection. Challenge of female mice with MOMP-specific IgG-opsonized C. muridarum delayed infection clearance but exacerbated oviduct occlusion. In male mice, MOMP-IgG elicited by immunization afforded no protection against testicular chlamydial infection, whereas; the transcytosis of IncA-IgG significantly reduced testicular chlamydial burden. Together these data show that the protective and pathological effects of IgG are dependent on FcRn-mediated transport as well as the specificity of IgG for intracellular or extracellular antigens.

Localization of glucocorticoid receptors at postsynaptic membranes in the lateral amygdala

Glucocorticoids, released in high concentrations from the adrenal cortex during stressful experiences, bind to glucocorticoid receptors in nuclear and peri-nuclear sites in neuronal somata. Their classically known mode of action is to induce gene promoter receptors to alter gene transcription. Nuclear glucocorticoid receptors are particularly dense in brain regions crucial for memory, including memory of stressful experiences, such as the hippocampus and amygdala. While it has been proposed that glucocorticoids may also act via membrane bound receptors, the existence of the latter remains controversial. Using electron microscopy, we found glucocorticoid receptors localized to non-genomic sites in rat lateral amygdala, glia processes, presynaptic terminals, neuronal dendrites, and dendritic spines including spine organelles and postsynaptic membrane densities. The lateral nucleus of the amygdala is a region specifically implicated in the formation of memories for stressful experiences. These newly observed glucocorticoid receptor immunoreactive sites were in addition to glucocorticoid receptor immunoreactive signals observed using electron and confocal microscopy in lateral amygdala principal neuron and GABA neuron soma and nuclei, cellular domains traditionally associated with glucocorticoid immunoreactivity. In lateral amygdala, glucocorticoid receptors are thus also localized to non-nuclear-membrane translocation sites, particularly dendritic spines, where they show an affinity for postsynaptic membrane densities, and may have a specialized role in modulating synaptic transmission plasticity related to fear and emotional memory.

Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

Feral pig populations are structured at fine spatial scales in tropical Queensland, Australia

Feral pigs occur throughout tropical far north Queensland, Australia and are a significant threat to biodiversity and World Heritage values, agriculture and are a vector of infectious diseases. One of the constraints on long-lasting, local eradication of feral pigs is the process of reinvasion into recently controlled areas. This study examined the population genetic structure of feral pigs in far north Queensland to identify the extent of movement and the scale at which demographically independent management units exist. Genetic analysis of 328 feral pigs from the Innisfail to Tully region of tropical Queensland was undertaken. Seven microsatellite loci were screened and Bayesian clustering methods used to infer population clusters. Sequence variation at the mitochondrial DNA control region was examined to identify pig breed. Significant population structure was identified in the study area at a scale of 25 to 35 km, corresponding to three demographically independent management units (MUs). Distinct natural or anthropogenic barriers were not found, but environmental features such as topography and land use appear to influence patterns of gene flow. Despite the strong, overall pattern of structure, some feral pigs clearly exhibited ancestry from a MU outside of that from which they were sampled indicating isolated long distance dispersal or translocation events. Furthermore, our results suggest that gene flow is restricted among pigs of domestic Asian and European origin and non-random mating influences management unit boundaries. We conclude that the three MUs identified in this study should be considered as operational units for feral pig control in far north Queensland. Within a MU, coordinated and simultaneous control is required across farms, rainforest areas and National Park Estates to prevent recolonisation from adjacent localities.

Stress effects caused by the expression of a mutant cellobiohydrolase I and proteasome inhibition in Trichoderma reesei Rut-C30

Trichoderma reesei Rut-C30 is used widely as an expression host for various gene products. We have explored cellular effects caused by the expression of a mutant form of cellobiohydrolase I (CBHI), the major secreted protein of T. reesei using biochemical and transcriptomic analyses and confocal laser scanning microscopy. The mutated CBHI was tagged fluorescently with Venus to establish the subcellular location of the fusion protein and its potential association with the proteasome, an organelle assigned for the disposal of misfolded proteins. Expression of the mutant CBHI in the high protein-secreting host Rut-C30 caused physiological changes in the fungal hyphae, affected protein secretion and elicited ER stress. A massive upregulation of UPR- and ERAD-related genes sec61, der1, uba1, bip1, pdi1, prp1, cxl1 and lhs1 was observed by qRT-PCR in the CBHIΔ4-Venus strain with four mutations introduced in the DNA encoding the core domain of CBHI. Further stress was applied to this strain by inhibiting function of the proteasome with MG132 (N-benzoylcarbonyl(Cbz)-Leu-Leu-leucinal). The effect of MG132 was found to be specific to the proteasome-associated genes. There are no earlier reports on the effect of proteasome inhibition on protein quality control in filamentous fungi. Confocal fluorescence microscopy studies suggested that the mutant CBHI accumulated in the ER and colocalized with the fungal proteasome. These results provide an indication that there is a limit to how far T. reesei Rut-C30, already under secretion stress, can be pressed to produce higher protein yields.

Localization of fibronectin in the rat testis : effects of serum and dibutyryl cyclic AMP on fibronectin deposition by peritubular myoid cells in culture

The peritubular zone of the rat testis has an extensive extracellular matrix (ECM). Fibronectin (FN) is distributed primarily in the basal lamina of the seminiferous tubule boundary tissue and is synthesized by peritubular myoid cells. Several extracellular changes are mediated by growth factors and these changes occur at the time of hormone mediated testicular development, particularly in the peritubular zone. The effects of serum or dibutyryl cyclic AMP (cAMP) on FN production by the mesenchymal peritubular myoid cells were evaluated. Rats of various ages (10, 15, 20, 40 and 80 days) were employed for immunofluorescent localization of rat testicular FN in frozen sections. In all age groups tested, FN was primarily present in a broad layer around each seminiferous tubule, and blood vessel, and in variable distribution throughout the interstitial stroma. By day 20 there was no clear distinction in FN staining between the peritubular zone and the interstitial tissue. This indicates an involvement of FN in the ECM developments which occur in the peritubular zone of the testis at this time. The peritubular myoid cells were isolated from 20-22 day old rat testis and cultured on glass coverslips. These cells were grown to confluence with 10% fetal calf serum (FCS) in medium until day 4 and then subcultured to have secondary monocultures maintained with or without serum. By means of immunofluorescence and cytochemistry using avidin-biotin peroxidase complex it was observed that peritubular myoid cells were positive for FN and most of the FN was localized in the perinuclear region. Subcultured peritubular myoid cells maintained for 4 days in medium containing FCS developed an extensive interconnecting FN matrix. In the presence of 0.5 mM cAMP in culture, FN became localized along the filamentous process of peritubular myoid cells and more prominently in the areas of triangulated multi-cell aggregates as well as on the surface of the contracted small spherical cells. The addition of cAMP in the presence of FCS, also caused a noticeable change in the staining pattern; FN was detected along the filamentous process developing into a complex network of cells encased in an extensive matrix. It would appear that the translocation of FN in the cytoplasmic extensions of peritubular myoid cells may be a direct consequence of morphological changes associated with metabolic regulation of cAMP. This may also be related to the puberty associated development of in vivo changes in the ECM produced by peritubular myoid cells.

Strategic targeting of the PI3K–NFκB axis in cisplatin-resistant NSCLC

Chemoresistance is a major therapeutic challenge to overcome in NSCLC, in order to improve the current survival rates of <15% at 5 years. We and others have shown increased PI3K signaling in NSCLC to be associated with a more aggressive disease, and a poorer prognosis. In this study, targeted inhibition of three strategic points of the PI3K–NFκB axis was performed with the aim of exploiting vulnerabilities in cisplatin-resistant NSCLC cells. Cisplatin-resistant cell lines were previously generated through prolonged exposure to the drug. Expression of PI3K and NFκB pathway-related genes were compared between cisplatin-resistant cells and their matched parent cells using a gene expression array, qRT-PCR, DNA sequencing, western blot, and immunofluorescence. Targeted inhibition was performed using GDC-0980, a dual PI3K–mTOR inhibitor currently in Phase II clinical trials in NSCLC, and DHMEQ, an inhibitor of NFκB translocation which has been used extensively both in vitro and in vivo. Effects of the two inhibitors were assessed by BrdU proliferation assay and multiparameter viability assay. NFKBIA was shown to be 12-fold overexpressed in cisplatin-resistant cells, with no mutations present in exons 3, 4, or 5 of the gene. Corresponding overexpression of IκBα was also observed. Treatment with DHMEQ (but not GDC-0980) led to significantly enhanced effects on viability and proliferation in cisplatin-resistant cells compared with parent cells. We conclude that NFκB inhibition represents a more promising strategy than PI3K–mTOR inhibition for treatment in the chemoresistance setting in NSCLC.

Discovery of an archetypal protein transport system in bacterial outer membranes

Bacteria have mechanisms to export proteins for diverse purposes, including colonization of hosts and pathogenesis. A small number of archetypal bacterial secretion machines have been found in several groups of bacteria and mediate a fundamentally distinct secretion process. Perhaps erroneously, proteins called 'autotransporters' have long been thought to be one of these protein secretion systems. Mounting evidence suggests that autotransporters might be substrates to be secreted, not an autonomous transporter system. We have discovered a new translocation and assembly module (TAM) that promotes efficient secretion of autotransporters in proteobacteria. Functional analysis of the TAM in Citrobacter rodentium, Salmonella enterica and Escherichia coli showed that it consists of an Omp85-family protein, TamA, in the outer membrane and TamB in the inner membrane of diverse bacterial species. The discovery of the TAM provides a new target for the development of therapies to inhibit colonization by bacterial pathogens.

Phosphorylation of caveolin-1 is anti-apoptotic and promotes cell attachment during oxidative stress of kidney cells

Aims: Caveolin-1 (cav1) is reported to have both cell survival and pro-apoptotic characteristics. This may be explained by its localisation or phosphorylation in injured cells. This study investigated the role of cav1 in kidney cells of different nephron origin and developmental state after oxidative stress. Methods: Renal MCDK distal tubular, HK2 proximal tubular epithelial cells and HEK293T renal embryonic cells were treated with 1mM hydrogen peroxide. Apoptosis, loss of cell adhesion, and cell survival were compared with expression of cav1 in its non-phosphorylated and phosphorylated (p-cav1) forms. Cav1 was transfected into the HEK293T cells, or caveolae were disrupted with filipin or nystatin in HK2 cells, to investigate functions of cav1 and p-cav1. Results: Oxidative stress induced more apoptosis in HK2s than MDCKs (p<0.05). HK2s had lower endogenous cav1 and p-cav1 than MDCKs (p<0.05). Both cell lines had increased p-cav1, but not cav1, with oxidative stress. This increase was greatest in MDCKs (p<0.01). Cav1 was located mainly in the plasma membrane of untreated cells and translocated to the cytoplasm with oxidative stress in both cell lines, more so in MDCKs. Disruption of caveolae caused cytoplasmic translocation of cav1 in HK2s, but did not alter high levels of oxidative stress-induced apoptosis. When HEK293Ts lacking endogenous cav1 were transfected with cav1, oxidant-induced apoptosis and loss of cell adhesion was decreased (p<0.01), and p-cav1 was induced by treatment. Conclusion: Cav1 expression and localisation in kidney cells is not anti-apoptotic, but increased expression of p-cav1 may promote cell survival after oxidative stress. © 2008 Royal College of Pathologists of Australasia.

The effects of geochemical conditions on the establishment and growth of mangrove seedlings in acid sulfate soil environments

Acid sulfate soils (ASS) is a stress factor that is responsible for the failure of some mangrove restoration projects, including abandoned aquaculture ponds converted from mangrove ecosystems. Through experimental and field studies, this research provides a better understanding of the biogeochemistry of ASS disturbance and the response of mangrove seedlings (Rhizophoraceae) under high metal levels and acidic conditions. This study found that mangrove restorations under ASS disturbance can work but with lower numbers of survived seedlings. To prevent toxicity under high levels of metal, seedlings retained metals in their roots and sparingly distributed them into aerial parts with low mobility. The presence of high levels of potential acidity parameters would allow pyrite to oxidise, thus increasing metal levels and acidity, which in turn affected the survival and growth of the seedlings.

Centralspindlin and α-catenin regulate Rho signalling at the epithelial zonula adherens

The biological impact of Rho depends critically on the precise subcellular localization of its active, GTP-loaded form. This can potentially be determined by the balance between molecules that promote nucleotide exchange or GTP hydrolysis. However, how these activities may be coordinated is poorly understood. We now report a molecular pathway that achieves exactly this coordination at the epithelial zonula adherens. We identify an extramitotic activity of the centralspindlin complex, better understood as a cytokinetic regulator, which localizes to the interphase zonula adherens by interacting with the cadherin-associated protein, α-catenin. Centralspindlin recruits the RhoGEF, ECT2, to activate Rho and support junctional integrity through myosin IIA. Centralspindlin also inhibits the junctional localization of p190 B RhoGAP, which can inactivate Rho. Thus, a conserved molecular ensemble that governs Rho activation during cytokinesis is used in interphase cells to control the Rho GTPase cycle at the zonula adherens

Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis

Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.

Product stability and sequestration mechanisms in Solanum tuberosum engineered to biosynthesize high value ketocarotenoids

To produce commercially valuable ketocarotenoids in Solanum tuberosum, the 4, 4′ β-oxygenase (crtW) and 3, 3′ β-hydroxylase (crtZ) genes from Brevundimonas spp. have been expressed in the plant host under constitutive transcriptional control. The CRTW and CRTZ enzymes are capable of modifying endogenous plant carotenoids to form a range of hydroxylated and ketolated derivatives. The host (cv. Désirée) produced significant levels of nonendogenous carotenoid products in all tissues, but at the apparent expense of the economically critical metabolite, starch. Carotenoid levels increased in both wild-type and transgenic tubers following cold storage; however, stability during heat processing varied between compounds. Subcellular fractionation of leaf tissues revealed the presence of ketocarotenoids in thylakoid membranes, but not predominantly in the photosynthetic complexes. A dramatic increase in the carotenoid content of plastoglobuli was determined. These findings were corroborated by microscopic analysis of chloroplasts. In tuber tissues, esterified carotenoids, representing 13% of the total pigment found in wild-type extracts, were sequestered in plastoglobuli. In the transgenic tubers, this proportion increased to 45%, with esterified nonendogenous carotenoids in place of endogenous compounds. Conversely, nonesterified carotenoids in both wild-type and transgenic tuber tissues were associated with amyloplast membranes and starch granules.

EphB4 localises to the nucleus of prostate cancer cells

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor.

Practical guidelines for planning and implementing fauna translocations

A successful translocation involves many complex factors, including a genetically appropriate source population that can sustain harvest, social and governmental support, assessment of disease transmission risk and a release site with appropriately secure habitat that can support population establishment and persistance. This information is typically discussed during staturory approval processes and can take considerable time. However, following approval, for translocations of most fauna, the initial critical step involves the inherently stressful process of capture, holding, transportation and release. This process is unpredictable and novel, and is especially challenging for wild animals when they are confined in close proximity to conspecifics and humans. In contrast, captive-reared animals have to cope with the unfamiliar challenges of finding food and shelter, along with coping with competition and predation. Little has been written in the scientific literature about the translocation process. This is unsurprising because this process has usually been the realm of skilled practioners, often with animal husbandry backgrounds, rather than research scientists. Highly skilled intuition, observation and the translocation practioner's equivalent of a 'green thumb' often guides the way. However, theory and experimentation, particularly on the effects of stress, is available and this work is invaluable for a successful translocation. Here, we provide a brief description of the translocation process, and discussion of what stress is and how it can be managed. We then provide practical guidelines for the successful translocation of invertebrates, lizards, turtles, passerine birds, marsupials and bats, using examples from Australia and New Zealand.