In vitro and in vivo evaluation of insulin-producing beta TC6-F7 cells in microcapsules

In the present study, the insulin secretory capacity ofTC6-F7 cells in microcapsules was evaluated. The cell mass within capsules was found to expand in a three-dimensional fashion, in contrast to cells seeded on plates that grew as a monolayer. In invitro studies, both free and encapsulated cells were found to secreteinsulin in the absence of glucose, at 13.6 ± 1.1 and 14.5 ± 0.9 ng · 10⁶cells¹ · 60 min¹, respectively, withthe response rising to a maximum of 26.0 ± 0.8 and 31 ± 2.3 ng · 10⁶cells¹ · 60 min¹ in the presence of16.8 mM glucose. Encapsulated cells were able to produceCa²⁺ responses in the presence ofKCl (50 mM) and BAY K 8644 (100 µM). In in vivo studies,intraperitoneal transplantation of 3.0 ×10⁶ microencapsulated cellsinto mice (n = 5) withstreptozotocin-induced diabetes resulted in the restoration ofnormoglycemia up to 57 days. Insulin concentrations rose from 0.4 ± 0.1 ng/ml before the graft administration to 2.2 ± 0.8 ng/ml afterthe transplantation in the normoglycemic recipients. An oral glucosechallenge in transplant recipients demonstrated a flat glucoseresponse, suggesting extremely high glucose clearance rates. These datademonstrate the potential use of the immunoisolated -cell lines forthe treatment of diabetes.

     

Correlation between the number of adult male Pectinophora gossypiella (Saund.) (Lep., Gelechidae) catches on pheromone traps and the rate of infestation in fruiting bodies of cotton plants by young larvae in three regions of central Greece

In order to evaluate the influence of the number of catches in pheromone-baited traps on the percentage of larval infestation, six delta traps equipped with sex attractant were placed in each of three regions in Central Greece (Farkadona, Farsala, Almyros) in cotton fields from 20 June until 30 September 1995. The collection of fruiting bodies took place weekly and the counting of adults in the traps was carried out each day. The population fluctuation in all three regions was similar with their peak during the first weeks of August. In Farkadona the infestation level was low (1% at the first sampling of August) with a maximum of 9% in the last sampling of September. In Farsala and Almyros, the infestation level was already high (10% and above) in early August. There was a significant positive linear correlation between the number of moth catches and the infestation percentage from first- and second-stage larvae on the first (R = 0694) and second (R = 0.7399) boll-feeding generations.     

IL-18 is correlated with type-2 immune response in children with steroid sensitive nephrotic syndrome

Children with steroid sensitive nephrotic syndrome (SSNS) is thought to have dysregulated type-1/type-2 cytokine network. Interleukin (IL)-18 is a cytokine, which may enhance both type-1 and type-2 responses, depending on the cytokines milieu. This prospective study aimed to assess type-1/type-2 cytokine synthesis and production profile in different stages of SSNS and define the potent involvement of IL-18. Twenty-three children with SSNS, aged 2.5-14 years, were studied; 23/23 both in active stage before treatment initiation and in remission still on steroids; 15/23 in remission off steroids as well. Data were compared with those obtained from 25 age-matched controls. The following parameters were assessed: Basic T cell populations, percentages of CD3+/CD69+/IFN-gamma+ and CD3+/CD69+/IL-4+ T cells as well as serum levels of IFN-gamma, IL-2, IL-4, IL-13 and IL-18. No difference in IL-2 levels was found between nephrotic children of all disease stages and controls (p>0.05). Percentage of CD3+/CD69+/IL-4+ T cells and serum levels of IL-4, IL-13 and IL-18 were significantly higher in the active stage of SSNS compared with the remission stages and controls (p<0.05). On the contrary, percentage of CD3+/CD69+/IFN-gamma+ T cells as well as serum IFN-gamma were significantly lower during active disease stage compared with remission stages and controls (p<0.05). In children with SSNS, of all disease stages, serum levels of IL-18 were significantly correlated with both IL-4 and IL-13 (r=0.628 and p<0.0001, r=0.71 and p<0.0001, respectively). It seems that a type-2 cytokine synthesis and production pattern prevails in children with active SSNS and IL-18 expression is significantly correlated with this type-2 immune response.     

Endoparasitoid wasp bracovirus-mediated inhibition of hemolin function and lepidopteran host immunosuppression

Successful embryonic development of parasitoid wasps in lepidopteran hosts is achieved through co-injection of polydna viruses whose gene products are thought to target the immune responses of the host. One gene product of the endosymbiont bracovirus of the parasitic wasp Cotesia rubecula, CrV1, has been reported to inhibit the immune responses of its endoparasitized lepidopteran host through interference with the haematocyte cytoskeletal structure. Here we establish that CcV1, the Cotesia congregata bracovirus orthologue of CrV1, is also uptaken by lepidopteran haemocytes and haemocyte-like established cell lines, but we also report on a different function of CcV1, which is highly relevant to the inhibition of the host immune responses and is based on its direct interaction with the pattern recognition molecule hemolin. Recombinant CcV1 inhibits hemolin functions, such as lipopolysaccharide binding and bacterial agglutination as well as bacterial phagocytosis by haemocytes and haemocyte-like cell lines, producing functional phenotypes equivalent to those observed to arise from RNAi-based inhibition of hemolin gene expression. Finally, we show that CcV1 and hemolin colocalize on the membrane surface of hemolin-expressing cells, a finding suggesting that CcV1 may be uptaken by haemocytes and inhibit haemocyte function as a result of its interaction with membrane-anchored hemolin.     

Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli

G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.     

Crystal Structure of the Engineered Neutralizing Antibody M18 Complexed to Domain 4 of the Anthrax Protective Antigen

The virulence of Bacillus anthracis is critically dependent on the cytotoxic components of the anthrax toxin, lethal factor (LF) and edema factor (EF). LF and EF gain entry into host cells through interactions with the protective antigen (PA), which binds to host cellular receptors such as CMG2. Antibodies that neutralize PA have been shown to confer protection in animal models and are undergoing intense clinical development. A murine monoclonal antibody, 14B7, has been reported to interact with domain 4 of PA (PAD4) and block its binding to CMG2. More recently, the 14B7 antibody was used as the platform for the selection of very high affinity, single-chain antibodies that have tremendous potential as a combination anthrax prophylactic and treatment. Here, we report the high-resolution X-ray structures of three high-affinity, single-chain antibodies in the 14B7 family; 14B7 and two high-affinity variants 1H and M18. In addition, we present the first neutralizing antibody-PA structure, M18 in complex with PAD4 at 3.8 Å resolution. These structures provide insights into the mechanism of neutralization, and the effect of various mutations on antibody affinity, and enable a comparison between the binding of the M18 antibody and CMG2 with PAD4.     

Role of Dimerization in the Catalytic Properties of the Escherichia coli Disulfide Isomerase DsbC

The bacterial protein-disulfide isomerase DsbC is a homodimeric V-shaped enzyme that consists of a dimerization domain, two α-helical linkers, and two opposing thioredoxin fold catalytic domains. The functional significance of the two catalytic domains of DsbC is not well understood yet. We have engineered heterodimer-like DsbC derivatives covalently linked via (Gly₃-Ser) flexible linkers. We either inactivated one of the catalytic sites (CGYC), or entirely removed one of the catalytic domains while maintaining the putative binding area intact. Variants having a single active catalytic site display significant levels of isomerase activity. Furthermore, mDsbC[H45D]-dim[D53H], a DsbC variant lacking an entire catalytic domain but with an intact dimerization domain, also showed isomerase activity, albeit at lower levels. In addition, the absence of the catalytic domain allowed this protein to catalyze in vivo oxidation. Our results reveal that two catalytic domains in DsbC are not essential for disulfide bond isomerization and that a determining feature in isomerization is the availability of a substrate binding domain.Disulfide bonds are critical for the proper folding and structural stability of many exocytoplasmic proteins. The Dsb family of thiol:disulfide oxidoreductase enzymes catalyzes oxidative protein folding in the periplasm of Escherichia coli by means of two independent pathways (1–3). In the DsbA-DsbB oxidation pathway, DsbA, a very strong oxidant, catalyzes the formation of disulfide bonds on newly translocated proteins (4). The DsbA disulfide is rapidly recycled by DsbB, a membrane protein that transfers electrons from DsbA onto quinones (5–7). In the DsbC-DsbD isomerization pathway, non-native disulfides are reduced or rearranged by DsbC. DsbC is maintained in a reduced, catalytically active state via the transfer of electrons from the inner membrane protein DsbD that in turn accepts electrons from thioredoxin 1 and ultimately from NADPH (via thioredoxin reductase) within the cytoplasm (8, 9). Large kinetic barriers keep the oxidation and isomerization pathways isolated, preventing the establishment of a futile cycle of electron transfer. Accordingly, reactions between enzymes of the two pathways, for example the oxidation of DsbC by DsbB or the reduction of DsbA by DsbD, are 10³–10⁷-fold slower than the physiologically relevant DsbA-DsbB and DsbC-DsbD reactions (10). Nonetheless, the kinetic barrier between DsbB and DsbC can be breached by introducing mutations that result in structural changes in DsbC (11, 12).DsbC is a homodimer with each monomer comprising an N-terminal dimerization domain and a C-terminal thioredoxin-like catalytic domain fused by an α-helical linker. The crystal structure of DsbC reveals that the two monomers come together to form a V-shaped protein. The inner surface of the resulting cleft is patched with uncharged and hydrophobic residues suggesting an important role in the binding of substrate proteins. The active sites comprising the sequence Cys⁹⁸-Gly⁹⁹-Tyr¹⁰⁰-Cys¹⁰¹ in each of the monomeric subunits are located in the arms of the “V” facing each other (13). Isomerization involves an attack onto a substrate disulfide by Cys⁹⁸ resulting in the formation of a mixed disulfide, which then is resolved by either another cysteine from the substrate or by Cys¹⁰¹ from DsbC (14, 15). Besides its isomerase activity, DsbC is also known to display chaperone activity preventing protein aggregation during refolding (16). In E. coli, disulfide bond isomerization is the limiting step in the oxidative folding of many heterologous proteins that contain multiple cysteines. Overexpression of DsbC has been shown to enhance the yield of proteins such as human nerve growth factor, human tissue plasminogen activator (tPA)² and immunoglobulins (17–19).DsbC is topologically analogous to the eukaryotic protein-disulfide isomerase (PDI). The structural similarities between the two enzymes may have resulted from convergent evolution by thioredoxin-like domain replication in the case of PDI and domain recruitment in DsbC (20, 21). PDI comprises two thioredoxin-like catalytic domains (a and a′) separated by two non-catalytic domains (b and b′), in addition to a c domain (22). In PDI, the catalytic domains are different and functionally nonequivalent (23). Substrate binding is mediated primarily by the b′ domain; the two catalytic domains, a and a′, can catalyze oxidation of small model peptides indicating that they must also have low substrate binding affinity (24).The DsbC monomer is essentially devoid of RNase A isomerase activity (25). Sun and Wang (44) reported that DsbC with one catalytic site impaired by carboxymethylation is also essentially inactive but, in separate studies, Zapun et al. (26) did not detect cooperativity between the two catalytic sites indicating that they function independently of each other (26). Moreover, unlike PDI, the significance of the putative peptide binding cleft of DsbC on disulfide isomerization has not been ascertained. However, while DsbA or TrxA with a PDI active site dipeptide (CGHC) display very little isomerase activity in vitro and in vivo (27–29), we recently showed that upon fusion to a dimerization region that provides a putative substrate binding surface (the E. coli peptidyl proline isomerase FkpA) they acquire the ability to assist the folding of periplasmically expressed multidisulfide heterologous proteins (30).In the present work, we engineered heterodimer-like covalently linked DsbC derivatives in which one of the catalytic sites has been inactivated (Fig. 1A) or one of the catalytic domains has been entirely removed while maintaining the intact peptide binding cleft (which is normally formed by association of the N-terminal domains of the two monomers) (Fig. 3A). We show that DsbC forced monomers with one functional active site, or with one thioredoxin domain only, display significant isomerization activity. Interestingly, the latter variant is partially reduced in vivo indicating that the presence of both thioredoxin domains is important for the avoidance of protein oxidation by DsbB.Open in a separate windowFIGURE 1.A, protein structure of DsbC, and molecular models of mDsbC-mDsbC and the single active site covalently linked mutants. Dimerization domains are shown in gray, thioredoxin domains in black, and the active sites in white. B, gel filtration FPLC of DsbC and linked variants. Purified proteins were run on a Superdex^TM 200 column in PBS, 10% glycerol buffer.Open in a separate windowFIGURE 3.A, molecular model of mDsbC-dim. Dimerization domains are shown in gray, thioredoxin domain in black, and catalytic site in white. B, gel filtration FPLC of mDsbC-dim as compared with DsbC. Purified proteins were run on a Superdex^TM 200 column in PBS, 10% glycerol buffer. C, MALS measurement of the molar masses of the components of mDsbC-dim together with their hydrodynamic radii. The data show monomeric, dimeric, and tetrameric states. The relative concentrations were determined by the refractive index differences.