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Objective: An imaging device to locate functionalised nanoparticles, whereby therapeutic agents are transported from the site of administration specifically to diseased tissues, remains a challenge for pharmaceutical research. Here, we show a new method based on electrical impedance tomography (EIT) to provide images of the location of gold nanoparticles (GNPs) and the excitation of GNPs with radio frequencies (RF) to change impedance permitting an estimation of their location in cell models \ud \ud Methods: We have created an imaging system using quantum cluster GNPs as contrast agent, activated with RF fields to heat the functionalized GNPs, which causes a change in impedance in the surrounding region. This change is then identified with EIT. \ud \ud Results: Images of impedance changes of around 80±4% are obtained for a sample of citrate stabilized GNPs in a solution of phosphate-buffered saline. A second quantification was carried out using colorectal cancer cells incubated with culture media, and the internalization of GNPs into the colorectal cancer cells was undertaken to compare them with the EIT images. When the cells were incubated with functionalised GNPs, the change was more apparent, approximately 40±2%. This change was reflected in the EIT image as the cell area was more clearly identifiable from the rest of the area. \ud \ud Significance: EIT can be used as a new method to locate functionalized GNPs in human cells and help in the development of GNP-based drugs in humans to improve their efficacy in the future.

International audience; Most pre-eclampsia (PE) studies have used cross-sectional data to derive conclusions regarding the pathophysiology of the condition. This has led to the concept that there exists early (34 weeks) disease according to gestational age at diagnosis. Survival time models have predicted that if the pregnancy was to continue indefinitely, all women would develop PE. In this study we have performed a longitudinal analysis of the urinary biomarker, inositol phosphoglycan (IPG), in a cohort of women giving birth in Mauritius (n-920). We have analysed the PE data in the traditional cross-sectional manner for n = 77 women who developed PE and also then looked at the longitudinal data for 71/77 of the same women. The data allows us to use longitudinal values to calculate a date of onset (first presence of biomarker in urine) and compare that to date of clinical diagnosis (cross sectional). We find two populations for both analysis consistent with an early and late stage subgroup. The calculated date of onset had subgroups (early and late) at 28.4 ± 0.41 weeks and 35.37 ± 0.26 weeks and for clinical date of diagnosis, 32.3 ± 0.59 weeks and 37.04 ± 0.62 weeks, respectively. The presence of the same biomarker in both subgroups and its ability to predict clinical onset 2–4 weeks prior to clinical diagnosis suggest that both groups may have similar aetiology.Urinary inositol phosphoglycan PPreeclampsia

Preeclampsia and eclampsia account for major pregnancy complications in Mauritius, an emerging country (maternal mortality rate of 60 per 100,000 deliveries). This prospective longitudinal study was carried out in the main regional hospital in the north of the island, to measure inositol phosphoglycan-P type (IPG-P) in the urine of pregnant women (using an ELISA-based assay). Women had approximately 10 prenatal visits per pregnancy and a complete follow-up in this same referral centre after the first trimester of pregnancy. Urine samples were collected every 1-4 weeks in all women. In a cohort of 416 patients, preeclampsia (PE) was diagnosed in 34 women. In established PE (hypertension and proteinuria), the assay as a diagnostic test showed a positive likelihood ratio of 18.73, a negligible negative likelihood ratio with area under the curve (AUC) of 0.99, sensitivity of 96.7%, specificity of 94.8% and remained negative in control women (n=312), women with gestational hypertension (without proteinuria (n=56), and gestational diabetic mothers (n=14). Moreover, as a predictive screening test two weeks before the diagnosis of PE, the assay showed sensitivity of 84.2% and specificity of 83.6%. Detection of urinary inositol phosphoglycan-P type in pregnant women can be a useful confirmatory marker of PE, as well as a predictive marker, two weeks before the onset of the disease.

Many plant-based systems have been developed as bioreactors to produce recombinant proteins. The choice of system for large-scale production depends on its intrinsic expression efficiency and its propensity for scale-up, post-harvest storage and downstream processing. Factors that must be considered include the anticipated production scale, the value and intended use of the product, the geographical production area, the proximity of processing facilities, intellectual property, safety and economics. It is also necessary to consider whether different species and organs affect the subcellular trafficking, structure and qualitative properties of recombinant proteins. In this article we discuss the subcellular localization and N-glycosylation of two commercially-relevant recombinant glycoproteins (Aspergillus niger phytase and anti-HIV antibody 2G12) produced in different plant species and organs. We augment existing data with novel results based on the expression of the sa me recombinant proteins in Arabidopsis and tobacco seeds, focusing on similarities and subtle differences in N-glycosylation that often reflect the subcellular trafficking route and final destination, as well as differences generated by unique enzyme activities in different species and tissues. We discuss the potential consequences of such modifications on the stability and activity of the recombinant glycoproteins.

A state of insulin resistance has been demonstrated in active preeclampsia, and women with clinical evidence of insulin resistance are at higher risk to develop this syndrome during pregnancy. Recently, inositol phosphoglycan P-type, a putative second messenger of insulin action, has been implicated in the pathophysiology of preeclampsia and is increased in the placenta, amniotic fluid, and maternal urine of preeclamptic women compared with normal pregnant women. We report here a case–control study to assess the potential of urinary levels of inositol phosphoglycan P-type as a screening test for preeclampsia. Twenty-seven preeclamptic women and 47 healthy pregnant women were recruited. A polyclonal antibody-based ELISA was developed to detect levels of inositol phosphoglycan P-type in urine. Its content in urinary specimens was found to be 30-fold higher in preeclamptic subjects than control subjects (329.1±21.8 versus 9.2±1.5; P 1 gestational date sample of urine was available, and retrospective analysis showed a significant time-related increase of the urinary level of inositol phosphoglycan P-type ≤7 weeks before clinical diagnosis of preeclampsia. Urinary level of inositol phosphoglycan P-type increased after diagnosis indicating a possible pathophysiological threshold level and steeply decreased after delivery.

Immunological alterations, endothelial dysfunction, and insulin resistance characterize preeclampsia. Endothelial cells hold the key role in the pathogenesis of this disease. The signaling pathways mediating these biological abnormalities converge on PKB/Akt, an intracellular kinase regulating cell survival, proliferation, and metabolism. Inositol second messengers are involved in metabolic and cell signaling pathways and are highly expressed during preeclampsia. Intracellular action of these molecules is deeply affected by zinc, manganese, and calcium. To evaluate the pathophysiological significance, we present the response of the intracellular pathways of inositol phosphoglycans involved in cellular metabolism and propose a link with the disease.

Adipose tissue plays a pivotal role in ageing and longevity; many studies, both human and animal, have focussed on the effects of food limitation. Here we present a new model based on striking differences between two 'normal' inbred strains of albino Wistar rats the Charles River (CR) and Harlan Olac (HO) that have marked differences in age-related accumulation of fat and insulin-stimulated rates of glucose incorporation into lipid in the epididymal fat pads (EFP). The incorporation [U-(14)C]glucose into lipid by adipocytes showed that the CR group had a twofold higher basal rate of lipogenesis and a greater response to insulin in vitro, exceptionally, adipocytes from CR group maintained the high response to insulin to late adulthood while retaining the lower EFP weight/100 g body weight. Inositol phosphoglycan A-type (IPG-A), a putative insulin second messenger, was 3.5-fold higher and cAMP significantly lower per EFP in the CR versus HO groups. Plasma insulin levels were similar and plasma leptin higher in CR versus HO groups. The anomaly of a higher rate of lipogenesis and response to insulin and lower EFP weight in the CR group is interpreted as the resultant effect of a faster turnover of lipid and stimulating effect of leptin in raising fatty acid oxidation by muscle, potentially key to the lower accumulation of visceral fat. The metabolic profile of the CR strain provides a template that could be central to therapies that may lead to the lowering of both adipose and non-adipocyte lipid accumulation in humans in ageing.

In preeclampsia, there is exacerbation of physiological changes associated with pregnancy such as insulin resistance, altered immune responses and inflammatory pathway activation. These exaggerated responses seen in preeclampsia are reminiscent of metabolic syndrome, and also are evident in gestational diabetes mellitus. The link between these phenomena is not clear but novel findings providing some insight have been reported recently. Inositol phosphoglycan P-type (P-IPG) in preeclampsia has been extensively investigated and increased production has been demonstrated. This molecule acts as a second messenger of insulin, enhances the metabolic effects of insulin and is associated with insulin resistance. This review article summarizes current evidence of the role of inositol phosphoglycans in the metabolic syndrome that occurs in preeclampsia, discussed in the light of modifications found in gestational diabetes mellitus and diabetes type 2 in pregnancy in humans and animal models. An increase in urinary release of P-IPG during pregnancy may herald the onset of preeclampsia. Further knowledge about the nature of the metabolic syndrome during preeclampsia and the degree of association between its components will help to inform future research efforts and to identify biochemical markers that could help in clinical practice, for example early markers that will have utility in managing disease progression.

Preeclampsia is a severe complication of human pregnancy and an insulin resistant state has been demonstrated in this multisystem disorder, although its bases remain unclear. Inositol phosphoglycans P-type belongs to a family of putative insulin mediators and was described to exert many insulin-like effects on lipid and glucose metabolism. A definite association between this molecule and preeclampsia was reported. The systemic inflammatory activation that occurs in preeclampsia as a consequence of the immunological dysfunction can exacerbate placental insulin resistance leading to an over-expression of P-IPG as a counterregulatory mechanism to insulin resistance. Besides, the lipidic form of P-IPG was reported to be similar to endotoxins, and may represent the link between insulin resistance, systemic inflammation and increased angiogenic factors. In this article we propose a new working theory on insulin resistance and preeclampsia.

Differences in biochemical and hormone profiles between two strains of rats provide insights into the relationships between insulin response, inositol phosphoglycans and lipid metabolism in adipose tissue. The results suggest the apparent anomaly of a higher rate of lipogenesis and response to insulin with a lower fat pad weight in the Charles River vs. Harlan Olac group relates to: (i) enzyme pre-programming with IPG-A, (ii) faster turnover of lipid, (iii) effects of leptin and cAMP.

A series of small-molecule microbicides has been developed for vaginal delivery to prevent heterosexual HIV transmission, but results from human clinical trials have been disappointing. Protein-based microbicides, such as HIV-specific monoclonal antibodies, have been considered as an alternative approach. Despite their promising safety profile and efficacy, the major drawback of such molecules is the economy of large-scale production in mammalian cells, the current system of choice. Here, we show that an alternative biomanufacturing platform is now available for one of the most promising anti-HIV antibodies (2G12). Our data show that the HIV-neutralization capability of the antibody is equal to or superior to that of the same antibody produced in CHO cells. We conclude that this protein production system may provide a means to achieve microbicide ingredient manufacture at costs that would allow product introduction and manufacture in the developing world.

Antibody 2G12 is one of a small number of human immunoglobulin G (IgG) monoclonal antibodies exhibiting potent and broad human immunodeficiency virus-1 (HIV-1)-neutralizing activity in vitro, and the ability to prevent HIV-1 infection in animal models. It could be used to treat or prevent HIV-1 infection in humans, although to be effective it would need to be produced on a very large scale. We have therefore expressed this antibody in maize, which could facilitate inexpensive, large-scale production. The antibody was expressed in the endosperm, together with the fluorescent marker protein Discosoma red fluorescent protein (DsRed), which helps to identify antibody-expressing lines and trace transgenic offspring when bred into elite maize germplasm. To achieve accumulation in storage organelles derived from the endomembrane system, a KDEL signal was added to both antibody chains. Immunofluorescence and electron microscopy confirmed the accumulation of the antibody in zein bodies that bud from the endoplasmic reticulum. In agreement with this localization, N-glycans attached to the heavy chain were mostly devoid of Golgi-specific modifications, such as fucose and xylose. Surprisingly, most of the glycans were trimmed extensively, indicating that a significant endoglycanase activity was present in maize endosperm. The specific antigen-binding function of the purified antibody was verified by surface plasmon resonance analysis, and in vitro cell assays demonstrated that the HIV-neutralizing properties of the maize-produced antibody were equivalent to or better than those of its Chinese hamster ovary cell-derived counterpart.

A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic N-glycosylation. A major concern is the presence of beta 1,2-xylose and core alpha 1,3-fucose residues on complex N-glycans as these nonmammalian N-glycan residues may provoke unwanted side effects in humans. In this study we have investigated the potential antigenicity of plant-type N-glycans attached to a human monoclonal antibody (2G12). Using glyco-engineered plant lines as expression hosts, four 2G12 glycoforms differing in the presence/absence of beta 1,2-xylose and core alpha 1,3-fucose were generated. Systemic immunization of rabbits with a xylose and fucose carrying 2G12 glycoform resulted in a humoral immune response to both N-glycan epitopes. Furthermore, IgE immunoblotting with sera derived from allergic patients revealed binding to plant-produced 2G12 carrying core alpha 1,3 fucosylated N-glycan structures. Our results provide evidence for the adverse potential of nonmammalian N-glycan modifications present on monoclonal antibodies produced in plants. This emphasizes the need for the use of glyco-engineered plants lacking any potentially antigenic N-glycan structures for the production of plant-derived recombinant proteins intended for parenteral human application.

An inappropriate glycogen accumulation in preeclamptic placentas was described as secondary to biochemical alterations. Insulin resistance is widely accepted to be associated with preeclampsia, although its basis remain unclear. A family of putative insulin mediators, namely inositol phosphoglycans, were described to exert many insulin-like effects on lipid and glucose metabolism. A definite association between the P-type mediator (P-IPG) and preeclampsia was reported, being increased in placenta, urine, amniotic fluid and cord blood from human preeclamptic pregnancies. A strong link exists between insulin resistance and inflammation. Clear features of insulin resistance and systemic inflammatory activation were described in preeclampsia. It may be a consequence of the immunological dysfunction that occurs in preeclampsia that is temporized during sperm exposure and co-habitation which confuses the maternal immune network to perceive 'danger'. The over-expression of P-IPG during preeclampsia may be a counter-regulatory mechanism to insulin resistance since these molecules mimic insulin action. Besides, the lipidic form of P-IPG was reported to be similar to endotoxins, and may represent the 'danger signa'. We propose here a novel working theory on insulin resistance and preeclampsia.

In recent years, plants have become an attractive alternative for the production of recombinant proteins. However, their inability to perform authentic mammalian N-glycosylation may cause limitations for the production of therapeutics. A major concern is the presence of beta1,2-xylose and core alpha1,3-fucose residues on complex N-linked glycans, as these N-glycan epitopes are immunogenic in mammals. In our attempts towards the humanization of plant N-glycans, we have generated an Arabidopsis thaliana knockout line that synthesizes complex N-glycans lacking immunogenic xylose and fucose epitopes. Here, we report the expression of a monoclonal antibody in these glycan-engineered plants that carry a homogeneous mammalian-like complex N-glycan pattern without beta1,2-xylose and core alpha1,3-fucose. Plant and Chinese hamster ovary (CHO)-derived immunoglobulins (IgGs) exhibited no differences in electrophoretic mobility and enzyme-linked immunosorbent specificity assays. Our results demonstrate the feasibility of a knockout strategy for N-glycan engineering of plants towards mammalian-like structures, thus providing a significant improvement in the use of plants as an expression platform.

We report the production of an important human therapeutic antibody in plant cell suspension cultures and the functional analysis of that antibody, including a comparison with the same antibody produced in CHO cells. We established transgenic tobacco BY2 suspension cell cultures expressing the human monoclonal antibody 2F5, which shows broadly neutralizing activity against HIV-1. The antibody was directed to the endoplasmic reticulum of the plant cells and was isolated by cell disruption, followed by protein A chromatography. The plant- derived antibody was shown to be largely intact by SDS- PAGE and immunoblot. Antigen binding activity was investigated by electrophoretic mobility shift assay and quantitatively determined by ELISA and Biacore biosensor technology. Ligand binding properties were analyzed using the ectodomain of human Fc gamma RI for kinetic analysis. The plant- derived antibody showed similar kinetic properties and 89% of the binding capacity of its CHO-derived counterpart, but was only 33% as efficient in HIV- 1 neutralization assays. Our results show that plant suspension cultures can be used to produce human antibodies efficiently and that the analysis methods used in this study, including biosensor technology, provide useful functional data about antibody performance. This highlights important issues raised by the use of plant systems to produce human biologics. Sack, M., Paetz, A., Kunert, R., Bomble, M., Hesse, F., Stiegler, G., Fischer, R., Katinger, H., Stoeger, E., Rademacher, T. Functional analysis of the broadly neutralizing human anti-HIV-1 antibody 2F5 produced in transgenic BY-2 suspension cultures.

Background/Aims: The mechanisms underlying overgrowth of adipose tissue in fetuses of women with gestational diabetes mellitus (GDM) are generally unknown. Inositol phosphoglycan A-type (A-IPG), a putative second messenger of insulin, was reported to regulate lipogenesis in adipose tissue. IPGs have recently been shown to increase during normal pregnancy, in maternal and fetal compartments. Methods: 48 women with GDM and 23 healthy pregnant women were recruited for this cross-sectional study. Levels of A-IPG were assessed enzymatically in urinary specimens and correlated with clinical parameters. Results: A-IPG urinary release was lower in GDM patients (p < 0.01) and correlated positively with BMI (p < 0.01) and negatively with glycaemic control in the diabetic group (postprandial glycaemia and glycated haemoglobin, p < 0.01) in addition to a nearly significant correlation with birth weight (p = 0.08). Furthermore, a lower A-IPG urinary release was found in diabetic subjects with normal fasting glycaemia compared with those with poor fasting glycaemic control (p < 0.05). Conclusions: An altered A-IPG urinary excretion occurs in GDM with a negative correlation with poor glycaemic control. Our data suggest an interesting potential role of this molecule in maternal metabolic control during pregnancy and, possibly, in fetal growth.

Plants have been established as an useful production system for commercially relevant proteins. Plant cell cultures show certain advantages compared to field-grown plants. However, one critical drawback for the long-term use of recombinant plant cell cultures is the instability of cell cultures concerning genetic background and productivity when maintained by subculturing. The ability to store recombinant cell lines stably by cryopreservation allows to maintain an efficient and stable production system. In this work, we describe the development of a cryopreservation protocol for a transgenic BY-2 cell culture expressing human serum albumin. In 1.75-L stirred-tank bioreactors growth and production kinetics of the transgenic cell line were compared after cryopreservation to the hitherto performed maintenance by subculturing. Growth and productivity of the cryopreserved cell culture remained stable after freezing for one week. Here, we show that we developed an efficient method which allows the storage of transgenic plant cell cultures, an important requirement for industrial processes.

Objectives Abnormal secretion of P-type inositol phosphoglycans (IPG-P) has been described in maternal urine of pre-eclamptic women. The aim of this study was to determine the origin of production of IPG-P. We examined the IPG-P content of maternal and fetal serum, maternal urine and amniotic fluid in both normal pregnancy and pre-eclampsia. Design Established extraction and bioactivity assay techniques were used to compare total IPG-P levels in serum samples, and a polyclonal-antibody-based ELISA to assay the amniotic fluid and urine samples in matched pairs of women. Subjects Eleven women with pre-eclampsia requiring caesarean section (subjects), 11 pregnant women requiring elective caesarean section for reasons other than pre-eclampsia (controls). Results Our data confirm the abnormal level of IPG-P in maternal urine during pre-eclampsia. Moreover, IPG-P levels were higher in umbilical sera than in maternal sera samples. Amniotic fluid as well as urine ELISA results were significantly higher in the pre-eclamptic group compared with normal controls. Total IPG-P bioactivity in serum did not vary between serum compartments in normal pregnancy. Uterine vein IPG-P levels were lower in pre-eclampsia when compared with normal pregnancy. A possible correlation was observed between urine and amniotic fluid levels in normal women. No correlation was observed between measured blood levels and those in urine and amniotic fluid. Conclusions It is hypothesized that steady state equilibrium of IPG-P in serum in normal pregnancy is disrupted in pre-eclampsia. Additionally, an abnormal IPG-P sub-fraction, detectable in urine and amniotic fluid, may be present and involved in the pathophysiology of the syndrome, although sites of production of this abnormal form remain unclear.

Many hormones such as insulin, insulin-like growth factors, and the glucocorticoids are involved in regulating fetal growth. Inositol phosphoglycans (IPGs), a family of putative second messengers of insulin, are reported to exert several of insulin's metabolic effects.A prospective cross-sectional study was carried out to investigate IPG P-type (P-IPG) in human amniotic fluid and in adult urine under physiological conditions. An amniotic fluid sample was taken from 78 women undergoing early amniocentesis and a mid-stream urine specimen was collected from 109 healthy pregnant and 66 non-pregnant women. All samples were assessed using a polyclonal antibody-based ELISA.The P-IPG content was a thousand times higher in the amniotic fluid than in the urine (p0.0001). Urinary specimens showed a four-fold higher P-IPG content during pregnancy than in healthy non-pregnant women (p0.001).Under physiological conditions, human amniotic fluid was found to be enriched in P-IPG compared with maternal urine, suggesting a possible fetal origin. Therefore, IPGs may play a role in insulin sensitivity and fetal growth and, perhaps, be involved in some of its abnormalities such as macrosomia and intrauterine growth restriction.

Glycosylphosphatidylinositol phospholipase D (GPI-PLD) has been proposed to be responsible for cleaving membrane-associated glycosylphosphatidyl inositol (GPI) molecules to generate inositol phosphoglycan (IPGs), which have growth factor-mimetic properties. We have cloned the mouse liver GPI-PLD cDNA, which has a sequence that differs from that previously isolated from a mouse glucagonoma cell library. Using a highly specific and very sensitive RNase protection assay, we found that the GPI-PLD expressed in adult/post-natal brain, antrum and insulin-producing cells is identical to that isolated from liver. The expression of mouse GPI-PLD in liver shows a complex genetic regulation with a mouse strain-specific variation. In addition, GPI-PLD mRNA levels were higher in 4-week old animals compared to older animals, and the GPI-PLD mRNA levels increased in mice that developed insulin dependent type 1 diabetes spontaneously. This suggests that the expression of liver GPI-PLD in mice is highly regulated.

The fixation of molecular O2 by the oxygenase activity of Rubisco leads to the formation of phosphoglycolate in the chloroplast that is further metabolized in the process of photorespiration. The initial step of this pathway is the oxidation of glycolate to glyoxylate. Whereas in higher plants this reaction takes place in peroxisomes and is dependent on oxygen as a co-factor, most algae oxidize glycolate in the mitochondria using organic co-factors. The identification and characterization of a novel glycolate dehydrogenase in Arabidopsis thaliana is reported here. The enzyme is dependent on organic co-factors and resembles algal glycolate dehydrogenases in its enzymatic properties. Mutants of E. coli incapable of glycolate oxidation can be complemented by overexpression of the Arabidopsis open reading frame. The corresponding RNA accumulates preferentially in illuminated leaves, but was also found in other tissues investigated. A fusion of the N-terminal part of the Arabidopsis glycolate dehydrogenase to red fluorescent protein accumulates in mitochondria when overexpressed in the homologous system. Based on these results it is proposed that the basic photorespiratory system of algae is conserved in higher plants.

Pre-eclampsia (PE) is a disorder affecting 5-10% of all pregnancies and is characterised by abnormal trophoblast invasion, maternal endothelial cell dysfunction and a systemic maternal response. A unifying factor responsible for eliciting these effects remains unknown. However, levels of the autocrine insulin mediators, inositolphosphoglycans (IPG), are elevated 3-fold in pre-eclamptic placentae compared with controls and are also elevated 3-fold in maternal urine of pre-eclamptic women, suggesting an abnormal paracrine role of the mediator in the systemic maternal response. At the placental level, IPGs are metabolic second messengers capable of eliciting some of the characteristic features of PE, such as the 10-fold increase in glycogen synthesis and 16-fold increase in the activity of the IPG-dependent enzyme glycogen synthase. IPGs are derived from their lipidic precursors, the glycosylphosphatidylinositols (GPI), in membrane associated caveolae by the action of a GPI-specific phospholipase D whose activity is regulated by its membrane microenvironment. We show that the lipidic GPI precursor was detected in total placental membrane and microvillous membrane from normal placentae. The presence of GPI could not be detected in PE placentae, suggesting that the GPI/IPG signalling system is dysregulated in this disorder. Equivalent amounts of a proteolytically-cleaved 50 kDa GPI-PLD protein is detected in both normal and PE placentae. However, GPI-PLD mRNA is absent, suggesting a mechanism of uptake from maternal serum. Since GPI-PLD, whose presence is required for hydrolysis of GPI and release of free IPG, is detectable with equal activity in both normal and PE placentae, we postulate that dysregulation of the tubular caveolar structure of the microvilli in pre-eclamptic placentae provides an environment which promotes the unregulated hydrolysis of GPI in this disorder.

Although plant biotechnology has been widely investigated for the production of clinical-grade monoclonal antibodies, no antibody products derived from transgenic plants have yet been approved by pharmaceutical regulators for clinical testing. In the Pharma-Planta project, the HIV-neutralizing human monoclonal antibody 2G12 was expressed in transgenic tobacco (Nicotiana tabacum). The scientific, technical and regulatory demands of good manufacturing practice (GMP) were addressed by comprehensive molecular characterization of the transgene locus, confirmation of genetic and phenotypic stability over several generations of transgenic plants, and by establishing standard operating procedures for the creation of a master seed bank, plant cultivation, harvest, initial processing, downstream processing and purification. The project developed specifications for the plant-derived antibody (P2G12) as an active pharmaceutical ingredient (API) based on (i) the guidelines for the manufacture of monoclonal antibodies in cell culture systems; (ii) the draft European Medicines Agency Points to Consider document on quality requirements for APIs produced in transgenic plants; and (iii) de novo guidelines developed with European national regulators. From the resulting process, a GMP manufacturing authorization was issued by the competent authority in Germany for transgenic plant-derived monoclonal antibodies for use in a phase I clinical evaluation. Following preclinical evaluation and ethical approval, a clinical trial application was accepted by the UK national pharmaceutical regulator. A first-in-human, double-blind, placebo-controlled, randomized, dose-escalation phase I safety study of a single vaginal administration of P2G12 was carried out in healthy female subjects. The successful completion of the clinical trial marks a significant milestone in the commercial development of plant-derived pharmaceutical proteins. The authors acknowledge funding from the European Union for the Pharma-Planta project (LSHB-CT-2003-503565) as well as additional funding for related and follow-on projects including CoMoFarm (227420), the COST Action Molecular Farming (FA0804), the Grand Challenges in Global Health and the Wellcome Trust (37872) and the ERC advanced grant FuturePharma (269110). We also would like to thank Fraunhofer Gesellschaft for strategic funding.

Phosphoenolpyruvate carboxylase (PEPC) plays a central role in the anaplerotic provision of carbon skeletons for amino acid biosynthesis in leaves of C3 plants. Furthermore, in both C4 and CAM plants photosynthetic isoforms are pivotal for the fixation of atmospheric CO2. Potato PEPC was mutated either by modifications of the N-terminal phosphorylation site or by an exchange of an internal cDNA segment for the homologous sequence of PEPC from the C4 plant Flaveria trinervia. Both modifications resulted in enzymes with lowered sensitivity to malate inhibition and an increased affinity for PEP. These effects were enhanced by a combination of both mutated sequences and pulse labelling with 14CO2 in vivo revealed clearly increased fixation into malate for this genotype. Activity levels correlated well with protein levels of the mutated PEPC. Constitutive overexpression of PEPC carrying both N-terminal and internal modifications strongly diminished plant growth and tuber yield. Metabolite analysis showed that carbon flow was re-directed from soluble sugars and starch to organic acids (malate) and amino acids, which increased four-fold compared with the wild type. The effects on leaf metabolism indicate that the engineered enzyme provides an optimised starting point for the installation of a C4-like photosynthetic pathway in C3 plants.

The enzyme glycosylphosphatidylinositol phospholipase D has a postulated role in the insulin-mimetic signaling pathway of glycosylphosphatidylinositol compounds. We have investigated enzyme activity in the serum of human type I diabetic patients and plasma and tissues of streptozotocin-induced diabetic rats following insulin administration. In the human diabetic patients serum enzyme activity fell by an average of 10.6% (SEM = 2.7; P = 0.008; n = 20) following administration of insulin. In addition serum enzyme activity appeared to be depleted by 27% (SEM = 8.8; P = 0.011; n = 10) compared to nondiabetic controls. In untreated diabetic rats plasma enzyme activity gradually increased 0.3-fold over a 6-week period (P < 0.001; n = 8), this increase was reversed and activity normalized when these animals were treated with insulin. Cloning of the rat glycosylphosphatidylinositol phospholipase D cDNA enabled confirmation of the liver as the principal organ of synthesis. Analysis of mRNA levels in the livers of the diabetic rats showed that gene expression was reduced in the insulin-treated animals compared to the noninsulin-treated controls by 0.7-fold (P = 0.004; n = 4). Tissue enzyme activity was also reduced in the insulin-treated rats; in skeletal muscle enzyme activity was 0.3-fold lower (P = 0.001; n = 4). Insulin therefore decreases glycosylphosphatidylinositol phospholipase D synthesis in diabetic animals resulting in decreased serum enzyme levels, suggesting a relationship between this enzyme and the function of insulin.

SummaryA number of immunomodulatory molecules are present in the placenta, including cytokines, prostaglandins, progesterone and indoleamine 2,3-dioxygenase. An undefined factor capable of down-regulating T-cell activity has recently been reported [1] as being produced by short-term cultures of placental fragments. By careful repetition of these studies we have confirmed that chorionic villi isolated from term placenta produce a low molecular weight, heat stable factor capable of inhibiting the IL-2-dependent proliferation of mouse CTLL-2 cells. This activity was not due, however, to a previously unknown immunosuppressive molecule, but rather to prostaglandin E2 (PGE2). Expression of cyclooxygenase (COX)-2 was detected in the syncytiotrophoblast of chorionic villi explants using immunohistochemistry. Culture of the explants in the presence of the COX-1/COX–2 inhibitors indomethacin and diclofenac, or with the COX-2-selective inhibitor DFP, blocked the production of the immunosuppressive factor. The immunosuppressive activity was restored by adding PGE2 to the supernatants obtained from diclofenac-inhibited explants. A number of different receptors are involved in mediating the biological effects of prostaglandins. By utilizing selective antagonists of individual receptors, we have established that the immunosuppressive effect of PGE2 on CTLL-2 cells is exerted via the EP4 receptor. Thus, addition of an EP4-selective antagonist, but not of EP1 or EP3 antagonists, abolished the immunosuppressive effect of PGE2 on CTLL-2 cells. This may have implications for attempts to selectively manipulate T-cell responses.

We have previously shown that infection with Plasmodium yoelii malaria or injection of extracts from malaria-parasitized red cells induces hypoglycemia in normal mice and normalizes the hyperglycemia in mice made moderately diabetic with streptozotocin. Inositol phosphoglycans (IPGs) are released outside cells by hydrolysis of membrane-bound glycosylphosphatidylinositols (GPIs), and act as second messengers mediating insulin action. The C57BL/Ks-db/db and C57BL/6J-ob/ob mice offer good models for studies on human obesity and Type 2 diabetes. In the present study, we show that a single iv injection of IPG-A or IPG-P extracted from P. yoelii significantly (P < 0.02) lowers the blood glucose in STZ-diabetic, db/db, and in ob/ob mice for at least 4--6 h. Using rat white adipocytes, IPG-P increased lipogenesis by 20--30% in the presence and absence of maximal concentrations of insulin (10(-8) M) (P < 0.01) and stimulated pyruvate dehydrogenase (PDH) phosphatase in a dose-related manner. Both IPG-A and IPG-P inhibited c-AMP-dependent protein kinase (PKA) in a dose-related manner. Compositional analysis of IPGs after 24 h hydrolysis revealed the presence of myo-inositol, phosphorus, galactosamine, glucosamine, and glucose in both IPG-A and IPG-P. However, hydrolysis of IPGs for 4 h highlighted differences between IPG-A and IPG-P. There are some functional similarities between P. yoelii IPGs and those previously described for mammalian liver. However, this is the first report of the hypoglycemic effect of IPGs in murine models of Type 2 diabetes. We suggest that IPGs isolated from P. yoelii, when fully characterized, may provide structural information for the synthesis of new drugs for the management of diabetes mellitus.

Here we report the structure of the human glycosylphosphatidylinositol phospholipase D1 gene, which covers at least 80 kb on chromosome 6p22. The gene comprises 25 exons and encodes a 5.8 kb mRNA, which was detected only in the liver. Southern blot analysis shows that the human genome contains only one GPLD gene and we could only detect one of the two previously reported cDNAs.

The ratio of two families of inositol phosphoglycans (IPG-A:IPG-P), insulin second messengers, is raised in non-insulin-dependent diabetes mellitus (NIDDM) and obesity. It is shown here that IPG A type inhibits leptin release from adipocytes, contrasting with the action of insulin (stimulation) and IPG P type (no effect). The significance of inhibitory effects of IPG A type on leptin release is important in relation to obesity and NIDDM in view of the action of leptin in promoting Lep expression and fat oxidation in muscle, in addition to its effects on satiety. Energy conservation and oxidation via interorgan regulation by leptin could be compromised by a rise in the IPG-A:IPG-P ratio.

Measurements have been made of the urinary content of inositol phosphoglycans IPG P-type and IPG A-type, putative insulin second messengers, in preeclampsia, in type I insulin-treated diabetic pregnant women and their matched control subjects, and nonpregnant women of child-bearing age. The content of IPG P-type and IPG A-type was also measured in the placenta from preeclamptic patients and from normal pregnancies. Pregnancy was associated with an increase, approximately twofold, in urinary output of IPG-P-type relative to nonpregnant controls (P

A progressive insulin resistant state develops throughout human pregnancy. Inositol phosphoglycan P-type (P-IPG), a second messenger of insulin, was reported to negatively correlate with the degree of insulin resistance in non-pregnant diabetic subjects. Urinary levels of P-IPG were assessed in insulin resistant states during pregnancy such as gestational diabetes mellitus (GDM, n=44) and type 2 diabetes mellitus (type 2 DM, n=25) and in 69 normal pregnant women. Urinary levels of P-IPG were higher in GDM than controls with a positive trend of release throughout normal pregnancy (P

The cDNA of the NADP-dependent malic enzyme gene Me 2 from the C 3 plant Flaveria pringlei was used for expression in Escherichia coli and Solanum tuberosum . A chimeric GST-Me 2 gene complemented a malic enzyme deficient E. coli mutant. Two potato lines were transformed with Me 2-cDNA constructs, one line already overexpressing the phospho enol pyruvate carboxylase gene ( ppc ) from Corynebacterium glutamicum . Both genes were under the control of the constitutive 35S CaMV promoter. Increased levels of malic enzyme (ME) were found in chloroplasts of transformants. Western blot analysis indicated that the ME transit sequence was cleaved. Expression of both genes led to a significantly reduced electron requirement for apparent CO 2 assimilation (e/A) at higher temperature. At low temperatures (15°C) 11 electrons per CO 2 assimilated (e/A) were measured in controls, single transformants ( ppc or Me 2) and double transformants ( ppc and Me 2). However, when leaf temperature was raised to 36°C electron requirement of the double transformants (15 e/A) was 65% of controls or single transformants (23 e/A). Thus, the temperature dependent increase in electron requirement was reduced in the double transformants suggesting a suppression in the oxygenation reaction of Rubisco and with it presumably in the rate of photorespiratory CO 2 release which is more marked at high light and high temperatures.

Glycoconjugate chemistry and glycobiology were rapidly evolving scientific disciplines in the 1980s. Their impact, however, on understanding fundamental biological processes was not immediately forthcoming. Within the past 12–18 moths, there has been a resurgence in the field with major discoveries leading to powerful new insights into the complex role of glycoconjugates in biological processes.

Extracts of human liver were found to contain activities which copurified and coeluted with the two major subtypes of mediators (type A and type P) isolated from insulin-stimulated rat liver. The putative type A mediator from human liver inhibited cAMP-dependent protein kinase from bovine heart, decreased phosphoenolpyruvate carboxykinase mRNA levels in rat hepatoma cells, and stimulated lipogenesis in rat adipocytes. The putative type P mediator stimulated bovine heart pyruvate dehydrogenase phosphatase. Both fractions were able to stimulate proliferation of EGFR T17 fibroblasts and the type A was able to support growth in organotypic cultures of chicken embryo cochleovestibular ganglia. Both activities were resistant to Pronase treatment and the presence of carbohydrates, phosphate, and free-amino groups were confirmed in the two fractions. These properties are consistent with the structure/function characteristics of the type A and P inositolphosphoglycans (IPG) previously characterized from rat liver. Further, the ability of the human-derived mediators to interact with rat adipocytes and bovine-derived metabolic enzymes suggests similarity in structure between the mediators purified from different species. Galactose oxidase-susceptible membrane- associated glycosylphosphatidylinositols (GPI) have been proposed to be the precursors of IPG. GPI was purified from human liver membranes followed by treatment with galactose oxidase and reduction with NaB3H4. Serial t.l.c. revealed three radiolabeled bands which comigrated with the putative GPI precursors found in rat liver. These galactose-oxidase-reactive lipidic compounds, however, were only partially susceptible to hydrolysis with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis and were resistant to glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei. These data indicate that IPG molecules with insulin-like biological activities are present in human liver., This work was supported by the Acción Integrada Hispano-Británica, by grants from the Medical Research Council, the Jules Thorn Trust, and the British Council to T.W.R. and Dirección General de Investigación, Ciencia y Tecnologı́a (DGICYT), Comunidad de Madrid, and Europharma (Boehringer Ingelheim group) to I.V-N. H.N.C. and S.K. are supported by the Medical Research Council (UK). Y.L. and M.A.A. are supported by Ministerio de Educación y Ciencia (Spain) and D.R.J. by The Royal Society (UK) and the Consejo Superior de Investigaciones Cientı́ficas (Spain).

Objective. This study examined the effect of exogenous dehydroepiandrosterone (DHEA) on the onset, incidence, and severity of collagen-induced arthritis (CIA). Methods. DHEA was administered subcutaneously prior to arthritis induction in DBA/1 mice, and the severity of the subsequent arthritis was monitored. Serum levels of total IgG and IgG isotype-specific anti-murine type II collagen were measured. Results. Repeated administration of DHEA during arthritis induction delayed the onset and decreased the severity of arthritis in male and female DBA/1 mice. DHEA failed to have an observable effect on established arthritis. IgG isotype autoantibody levels were found to be decreased in the sera of DHEA-treated mice. Conclusion. Administration of exogenous DHEA offered protection against the development of CIA. These data support the results of human studies in which low DHEA levels have been identified as a potential risk factor for the development of rheumatoid arthritis. These findings also highlight DHEA as a potential therapy worthy of further investigation.

Phosphoenolpyruvate carboxylase (PEPC) genes from Corynebacterium glutamicum (cppc), Escherichia coli (eppc) or Flaveria trinervia (fppc) were transferred to Solanum tuberosum. Plant regenerants producing foreign PEPC were identified by Western blot analysis. Maximum PEPC activities measured in eppc and fppc plants grown in the greenhouse were doubled compared to control plants. For cppc a transgenic plant line could be selected which exhibited a fourfold increase in PEPC activity. In the presence of acetyl-CoA, a known activator of the procaryotic PEPC, a sixfold higher activity level was observed. In cppc plants grown in axenic culture PEPC activities were even higher. There was a 6-fold or 12-fold increase in the PEPC activities compared to the controls measured in the absence or presence of acetyl-CoA, respectively. Comparable results were obtained by transient expression in Nicotiana tabacum protoplasts. PEPC of C. glutamicum (PEPC C.g.) in S. tuberosum leaf extracts displays its characteristic K(m) (PEP) value. Plant growth was examined with plants showing high expression of PEPC and, moreover, with a plant cell line expressing an antisense S. tuberosum (anti-sppc) gene. In axenic culture the growth rate of a cppc plant cell line was appreciably diminished, whereas growth rates of an anti-sppc line were similar or slightly higher than in controls. Malate levels were increased in cppc plants and decreased in antisense plants. There were no significant differences in photosynthetic electron transport or steady state CO2 assimilation between control plants and transformants overexpressing PEPC C.g. or anti-sppc plants. However, a prolonged dark treatment resulted in a delayed induction of photosynthetic electron transport in plants with less PEPC. Rates of CO2 release in the dark determined after a 45 min illumination period at a high proton flux density were considerably enhanced in cppc plants and slightly diminished in anti-sppc plants. When CO2 assimilation rates were corrected for estimated rates of mitochondrial respiration in the light, the electron requirement for CO2 assimilation determined in low CO2 was slightly lower in transformants with higher PEPC, whereas transformants with decreased PEPC exhibited an appreciably elevated electron requirement. The CO2 compensation point remained unchanged in plants (cppc) with high PEPC activity, but might be increased in an antisense plant cell line. Stomatal opening was delayed in antisense plants, but was accelerated in plants overexpressing PEPC C.g. compared to the controls.

Metabolic alterations occur along with immunological alterations, abnormal placental development and endothelial cell dysfunction in preeclampsia. Typical features of placental alterations are an inadequate/incomplete trophoblast invasion of maternal spiral arteries and acute atherosis. Phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway holds a pivotal role in intracellular metabolic pathways as it regulates endothelial cell survival, migration, tube formation and nitric oxide production. The inositol second messenger system (myo- inositol and D-chiro inositol) is involved in metabolic and cell signaling pathways. Pyruvate dehydrogenase complex (PDC) is at the interface between glycolysis and the citric acid cycle and is crucial to the generation of ATP, acetyl CoA and NADH by mitochondria. We showed that the putative insulin mediator inositol phosphoglycan containing D-chiro inositol (IPG-P) has a sigmoidal inhibitory action on PDK in addition to its known linear stimulation of PDP. Thus, at critical levels of these mediators this sigmoidal/linear model amplifies the switchover from the inactive to active form of PDC, a ‘push- pull’ system that combined with the hormonal control of IPG-P, indicates a powerful regulatory function via inhibition of PDK. The presence of zinc is of interest in relation to our reported effects of zinc ions on PDP and PDH kinases, and in the context of the cardioprotective effects of zinc on vascular ischemia-reperfusion injury. This resetting of the PDC to the active non-phosphorylated state and formation of acetyl-CoA may be enhanced by the effect of zinc on the activation of enolase in the glycolytic pathway and by the regulation of the phosphorylation state of the insulin receptor and thus downstream pathways favouring the provision of pyruvate from glucose. The counter effect of zinc in depressing acetyl-CoA oxidation is obtained via inhibition of key enzymes of the tricarboxylic acid cycle driving acetyl-CoA towards the lipogenic route. Manganese is present in high concentrations in IPG-A type (containing myo-inositol) and coordinates lipid synthesis via manganese activation of different enzymes. Characterization of the intracellular action of inositol messengers in the context of a metabolic disease like preeclampsia helps to understand the underlying biochemical mechanisms that occur in placenta and endothelial cells.

The glycosylation of IgG results in many different glycoforms. A large body of correlative data (including remission of arthritis during pregnancy) has suggested that IgG molecules lacking galactose were associated with rheumatoid arthritis. We now demonstrate that agalactosyl IgG glycoforms are directly associated with pathogenicity in murine collagen-induced arthritis. We show that passive transfer of an acute synovitis in T-cell-primed mice can be enhanced by using IgG containing autoantibodies to type II collagen when the antibodies are present as the agalactosyl glycoform. Thus, nonpathogenic doses of autoantibodies can be made pathogenic by altering their glycosylation state.

Immunosuppressive factors isolated from trophoblast are known to block both innate and major histocompatibility complex (MHC)-dependent cell-mediated immune responses in vitro and, in some cases, in vivo. We investigated the biochemical nature of these factors, which is presently unknown. Immunosuppressive activity, assessed by inhibition of two-way MLR, was extracted from term syncytiotrophoblast microvilli using 3 M KCl. The activity resisted both extensive pronase digestion and heating to 90 degrees C for 1 h, demonstrating that intact membrane proteins were not required. Although purified protein-linked oligosaccharides released by hydrazinolysis from the syncytiotrophoblast membrane were themselves inactive, they blocked the immunosuppressive activity of the KCl extract. After pronase digestion, the activity could be fractionated by TSK 55S gel filtration, followed by C18 reverse-phase chromatography. Sequential exoglycosidase digestion of hydrazine-released sugars of the active fraction demonstrated that it contained neutral N-linked oligomannose and hybrid oligosaccharides, which normally make up < 3% of the total syncytiotrophoblast-derived protein glycan library. These glycopeptides of the active fraction were associated with membrane phospholipid micelles. The possible mechanism by which incompletely processed N-linked oligosaccharides expressed by a variety of syncytiotrophoblast membrane glycoproteins may block allogeneic reactivity when presented as polyvalent sugar groups is discussed.

International audience; BACKGROUND: Inositol phosphoglycan P-type (P-IPG) has consistently found to be elevated during active preeclampsia, although the biosynthetic source has to be identified yet. This multicenter prospective cross-sectional case-control study evaluated the fetus/newborn as the source of P-IPG. METHODS: A urine specimen was collected longitudinally for three consecutive days after delivery from 90 newborns and their mothers, and ordered according to clinical diagnosis of preeclampsia, gestational hypertension, or healthy pregnancy. RESULTS: The urinary excretion of P-IPG on day 0 was higher in the mothers in all groups (p \textless 0.05) with higher levels in preeclamptic women (p \textless 0.01) in the mothers compared to their newborns in the preeclamptic group (p\textless0.01). The difference persisted at least two days post partum. CONCLUSION: Findings of this study confirm the specificity of the increase in urinary excretion of P-IPG in preeclamptic mothers at day of birth compared to healthy pregnancy and GH, but does not extend to their newborns.

Endometrial glands represent an important source of nutrients for the conceptus during the first trimester. Their secretions are enriched with carbohydrates, and glycogen accumulates within the syncytiotrophoblast of the placenta. It has been assumed that fetal and placental metabolism follow adult pathways, although it is now appreciated that early development occurs in a low-oxygen environment. In past decades, a novel family of putative insulin mediators, inositol phosphoglycans (IPGs), was discovered. These molecules act as allosteric activators and/or inhibitors of enzymes and transduction proteins involved in the control of cell signalling and metabolic pathways, and determine the specificity of responses after activation of the insulin receptor. One member, IPG P-type, activates pyruvate dehydrogenase phosphatase (PDH-Pase), glycogen synthase phosphatase, and glycerol-3-phosphate acyltransferase. Activation of key phosphatases play a major role in the regulation of glucose disposal by oxidative metabolism via PDH, and the non-oxidative storage by glycogen synthesis, both pathways classically known to be regulated by insulin. High concentrations of IPG P-type in amniotic fluid suggest a role in the regulation of carbohydrate metabolism in the fetal-placental unit. Glycogen accumulation in the syncytiotrophoblast also occurs in preeclamptic pregnancies, and is consistently associated with higher placental levels of IPG P-type. Here, we explore the relationship between nutrients provided by the endometrial glands during early pregnancy, IPG P-type and fetal metabolic requirements. We also discuss whether a disconnect between the placental/fetal metabolic state and oxygen tension could lead to a preeclamptic-type syndrome via leakage of Warburg/IPG mediators into the maternal circulation.

The metabolic syndrome that occurs in preeclampsia reflects the complex interactions between immunological alterations and the systemic inflammation that have been shown to take place during this complication of human pregnancy. Inositol phosphoglycans play a definite role in the insulin resistance in preeclampsia with a higher production and urinary excretion of this molecule before and during preeclampsia. Recent researches suggest that the feto-placental glucose metabolism in the first and early second trimester is mainly linked to the nonoxidative pathway of glycogen catabolism supporting the pivotal role of the inositol phosphoglycan P-type. In this article we present the results of a case-control study carried out in the first trimester to evaluate the potential of urinary P-IPG release as a early marker of the disease. A single mid-stream sample of maternal urine was collected at 11 weeks of gestation for this single centre retrospective study. Twenty-seven patients out of 331 women recruited (8.1%) went on to develop preeclampsia but no sample attained positivity. Further details about the development of the metabolic syndrome during preeclampsia were retrieved also from other studies to implement our knowledge about the pathophysiology of this syndrome and to identify biochemical aspects that could help in clinical practice.

Hypertensive disorders represent the most common complications of human pregnancy with substantial impact on fetal and maternal outcomes. Inositol phosphoglycan P-type has recently been identified as a novel marker of preeclampsia, the most severe form of hypertension during pregnancy, with a significant increase in urinary excretion preceding the clinical diagnosis.A prospective, longitudinal study was carried out to assess the potential of urinary levels of inositol phosphoglycan P-type as a screening test for preeclampsia. A specific ELISA-based test was used to assess urinary levels of P-IPG.Nine patients out of 93 women recruited (496 urinary samples were collected) went on to develop preeclampsia in a cohort of women with high-risk pregnancies. A cut-off value of urinary inositol phosphoglycan P-type was identified by ROC analysis providing a sensitivity and specificity for the current protocol of 88.9% and 62.7%, respectively. Twenty-three women with healthy pregnancies had sporadic episodes of increased excretion of inositol phosphoglycan P-type during pregnancy that consistently resolved back to normal baseline without development of preeclampsia. There was no correlation of urine levels of inositol phosphoglycan P-type and urine protein and patients with gestational hypertension had normal levels of urine inositol phosphoglycan P-type.These findings suggest that, given the rapid raise of P-IPG before the onset of the disease, multiple assessments may help at identifying women at risk of developing preeclampsia.

P>The use of vaginal microbicides containing human immunodeficiency virus (HIV)-neutralizing antibodies (nAbs) is a promising strategy to prevent HIV-1 infection. Although antibodies are predominantly manufactured using mammalian cells, elastin-like peptide (ELP) fusion technology improves the stability of recombinant, plant-produced proteins and facilitates their purification, making plants an alternative platform for antibody production. We generated transgenic tobacco plants accumulating four different formats of the anti-HIV-1 antibody 2G12 in the endoplasmic reticulum (ER), i.e. with ELP on either the light or heavy chain, on both, or on neither. Detailed analysis of affinity-purified antibodies by surface plasmon resonance spectroscopy showed that the kinetic binding parameters of all formats were identical to 2G12 lacking ELP produced in Chinese hamster ovary (CHO) cells. Importantly, protein purification from seeds by inverse transition cycling (ITC) did not affect the binding kinetics. Analysis of heavy chain N-glycans from leaf-derived antibodies showed that retrieval to the ER was efficient for all formats. In seeds, however, N-glycans on the naked antibody were extensively trimmed compared with those on the ELP fusion formats, and were localized to a different subcellular compartment. The in vitro HIV-neutralization properties of the tobacco-derived 2G12 were equivalent to or better than those of the CHO counterpart.

Among the many plant-based production systems that have been developed for pharmaceutical proteins, seeds have the useful advantage of accumulating proteins in a relatively small volume, and recombinant proteins are very stable in dry seeds allowing long-term storage and facilitating distribution before processing.To take full advantage of the natural ability of endosperm cells to store large amounts of protein in a protected subcellular environment, it is useful to target recombinant proteins to appropriate storage organelles. In this chapter, we describe the distinct types of protein storage organelles in the cereal endosperm and a protocol for the detection of recombinant proteins in these organelles by immunofluorescence and immunogold labelling.The use of food and feed crops for the production of pharmaceutical proteins such as edible vaccines implies the need for strict separation of the transgenic seeds from the food and feed chain. For improved traceability visual markers may be co-expressed with the gene of interest in engineered seeds. DsRed is one example for a fluorescent protein that can be detected with high sensitivity using low tech equipment. We therefore describe the generation of transgenic maize plants expressing DsRed in a constitutive manner, and we point out the advantages of using this marker during the process of transformation and selection of plant tissue and later during breeding of transgenic lines into elite germplasm.

Glycosylated lectins represent a series of glycoproteins with related activities and, in the case of the Leguminosae, related amino acid sequences. Therefore, they offer a model system in which to study the diversity of N-linked oligosaccharide structures of plant glycoproteins. The influence of the polypeptide on the type of oligosaccharide substitution and the problem of inter- and intra-genus variation in glycosylation can also be addressed. Analysis of the glycosylation of 18 lectins has shown that they can be classified into four qualitatively similar groups on the basis of the Bio-Gel P-4 elution profiles of the oligosaccharides released by hydrazinolysis: (a) The Erythrina cristagalli profile, with a major component at 8.8 glucose units (gu) and minor components at 8.0, 7.2, and 5.8 gu. The major component is the heptasaccharide, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-[beta-D-Xyl p-(1----2)]- beta-D-Manp-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp-(1----3)]- D-GlcNAc. (b) The Phaseolus vulgaris profile, which was characterized by peaks at 12.5, 11.7, 10.8, and 9.9 gu, in addition to the peaks at 8.8, 8.0, 7.2, and 5.8 gu mentioned above. These higher-mol.-wt. components were oligo-D-mannose oligosaccharides containing 9, 8, 7, and 6 D-mannose residues, respectively. (c) The Lonchocarpas capassa profile, which had a major peak at approximately 8 gu. (d) The soybean agglutinin profile, which has a single peak at 12.5 gu. This peak consisted solely of an oligomannose undecasaccharide containing 9 D-mannose residues. This lectin is unique in that it shows no microheterogeneity.

Abnormal metabolism of inositol phosphoglycan P-type (P-IPG) has been described in insulin-resistant states. Recently, a definite link between P-IPG and preeclampsia has been reported. P-IPG release after insulin stimulus has been described in the placental tissue of healthy women and a complete absence of P-IPG release has been found in preeclamptic samples, associated with disturbed insulin signaling. This study was undertaken to assess the release of this mediator in intrauterine growth restriction (IUGR) and hypertensive disorders other than preeclampsia.Seven women with IUGR, seven with gestational hypertension, 11 with preeclampsia, and 12 controls were recruited for this study. Fresh placental membranes were prepared and incubated with human recombinant insulin. Bioactivity of P-IPG released after insulin stimulus was assessed using a specific bioassay. A multiple comparison between groups was carried out. The study population provided a statistical power of 0.94.P-IPG release was highest and lowest from healthy and preeclamptic samples, respectively (p0.01). Specimens from patients with IUGR and gestational hypertension released less P-IPG than did controls (p0.05).Abnormal release of P-IPG from placentas of IUGR and gestational hypertensive mothers seems to confirm an association between these disorders of human pregnancy and insulin resistance.