Apparent Secretion Research Articles

Impact of the nisin modification machinery on the transport kinetics of NisT

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides containing dehydrated amino acids and (methyl-)lanthionine rings. One of the best-studied examples is nisin produced by Lactococcus lactis. Nisin is synthesized as a precursor peptide comprising of an N-terminal leader peptide and a C-terminal core peptide. Amongst others, the leader peptide is crucial for enzyme recognition and acts as a secretion signal for the ABC transporter NisT that secretes nisin in a proposed channeling mechanism. Here, we present an in vivo secretion analysis of this process in the presence and absence of the nisin maturation machinery, consisting of the dehydratase NisB and the cyclase NisC. Our determined apparent secretion rates of NisT show how NisB and NisC modulate the transport kinetics of NisA. Additional in vitro studies of the detergent-solubilized NisT revealed how these enzymes and the substrates again influence the activity of transporter. In summary, this study highlights the pivotal role of NisB for NisT in the secretion process.

Peptidoglycan Branched Stem Peptides Contribute to Streptococcus pneumoniae Virulence by Inhibiting Pneumolysin Release.

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and is a significant pathogen worldwide. Pneumolysin (Ply) is a multi-functional, extracellular virulence factor produced by this organism that is critical for pathogenesis. Despite the absence of any apparent secretion or cell surface attachment motifs, Ply localizes to the cell envelope of actively growing cells. We sought to characterize the consequences of this surface localization. Through functional assays with whole cells and subcellular fractions, we determined that Ply activity and its release into the extracellular environment are inhibited by peptidoglycan (PG) structure. The ability of PG to inhibit Ply release was dependent on the stem peptide composition of this macromolecule, which was manipulated by mutation of the murMN operon that encodes proteins responsible for branched stem peptide synthesis. Additionally, removal of choline-binding proteins from the cell surface significantly reduced Ply release to levels observed in a mutant with a high proportion of branched stem peptides suggesting a link between this structural feature and surface-associated choline-binding proteins involved in PG metabolism. Of clinical relevance, we also demonstrate that a hyperactive, mosaic murMN allele associated with penicillin resistance causes decreased Ply release with concomitant increases in the amount of branched stem peptides. Finally, using a murMN deletion mutant, we observed that increased Ply release is detrimental to virulence during a murine model of pneumonia. Taken together, our results reveal a novel role for branched stem peptides in pneumococcal pathogenesis and demonstrate the importance of controlled Ply release during infection. These results highlight the importance of PG composition in pathogenesis and may have broad implications for the diverse PG structures observed in other bacterial pathogens.

Midkine-a protein localization in the developing and adult retina of the zebrafish and its function during photoreceptor regeneration.

Midkine is a heparin binding growth factor with important functions in neuronal development and survival, but little is known about its function in the retina. Previous studies show that in the developing zebrafish, Midkine-a (Mdka) regulates cell cycle kinetics in retinal progenitors, and following injury to the adult zebrafish retina, mdka is strongly upregulated in Müller glia and the injury-induced photoreceptor progenitors. Here we provide the first data describing Mdka protein localization during different stages of retinal development and during the regeneration of photoreceptors in adults. We also experimentally test the role of Mdka during photoreceptor regeneration. The immuno-localization of Mdka reflects the complex spatiotemporal pattern of gene expression and also reveals the apparent secretion and extracellular trafficking of this protein. During embryonic retinal development the Mdka antibodies label all mitotically active cells, but at the onset of neuronal differentiation, immunostaining is also localized to the nascent inner plexiform layer. Starting at five days post fertilization through the juvenile stage, Mdka immunostaining labels the cytoplasm of horizontal cells and the overlying somata of rod photoreceptors. Double immunolabeling shows that in adult horizontal cells, Mdka co-localizes with markers of the Golgi complex. Together, these data are interpreted to show that Mdka is synthesized in horizontal cells and secreted into the outer nuclear layer. In adults, Mdka is also present in the end feet of Müller glia. Similar to mdka gene expression, Mdka in horizontal cells is regulated by circadian rhythms. After the light-induced death of photoreceptors, Mdka immuonolabeling is localized to Müller glia, the intrinsic stem cells of the zebrafish retina, and proliferating photoreceptor progenitors. Knockdown of Mdka during photoreceptor regeneration results in less proliferation and diminished regeneration of rod photoreceptors. These data suggest that during photoreceptor regeneration Mdka regulates aspects of injury-induced cell proliferation.

One-Stage Replacement Surgery of Orbital Implants With Noninfectious Complications

Orbital implant-associated complications always affect patients, resulting in multiple operations. One-stage replacement of complicated implants has been attractive for reducing operation times and preventing long-time appearance without artificial eye. We retrospectively analyzed the outcomes of 1-stage replacement surgery of orbital implant. A total of 21 eyes in 21 cases with noninfectious complications, who failed conservative therapies and surgical repairs, were treated with 1-stage orbital implant replacement. After a detailed ophthalmic history was obtained and examinations performed, patients with apparent purulent secretion in conjunctival sac were excluded. The preexisting orbital implant was carefully removed, and a new hydroxyapatite sphere was implanted simultaneously. Wound healing status, prosthesis movement, and cosmetic features were evaluated and recorded after at least 6 months' follow-up. Orbital implant exposure(16 cases) was the main indication of the replacement surgery; other causes included superior orbital area depression (2 cases), conjunctival fistula (1 case), orbital implant malposition (1 case), and the metal material of implant obstructing magnetic resonance imaging scan in 1 case. Eye-socket hollow was redressed in all cases immediately, and 95.23% cases (20/21) could wear an artificial eye and had a good cosmetic appearance, whereas conjunctival sac fistula reoccurred in 1 patient, and the implant was removed ultimately. Conjunctival sac constrictions occurred in 4 cases after operation and were treated by secondary conjunctival sac reconstruction 6 months later. In conclusion, 1-stage replacement of orbital implants offers an effective and efficient strategy for dealing with severe postimplantation noninfectious complications in achieving a symmetrically cosmetic appearance without delay.

Diarrhoeal disease through enterocyte secretion: a doctrine untroubled by proof

For almost 40 years, one of the principal causes of diarrhoeal disease has been thought to be fluid secretion emanating from the epithelial cells of the small and large intestine. Given the extremely large fluid losses seen in cholera, where secretion can be up to several litres per day, this seems a plausible hypothesis. The enterocyte (epithelial cell) secretion hypothesis rapidly displaced all other alternatives, such as vasodilatation coupled with enhanced paracellular permeability. An essential mechanism underlying enterocyte secretion has always been assumed to be electrogenic chloride secretion, leading to a localized osmotic imbalance at the mucosal surface of the enterocytes that causes fluid entry into the lumen by osmosis. The chloride secretion basis for enterotoxin-deranged secretion is assumed to be measurable by changes in electrical currents and by altered transport of chloride ion. These can be detected after the small intestine is exposed to a heat-stable enterotoxin (STa) produced by Escherichia coli. However, in vivo, when the recovered volume technique is used, STa is found not to be secretory. The heat-stable enterotoxin is therefore a test case toxin, because the complex techniques used to demonstrate enterocyte secretion after STa exposure show apparent secretion, while the simplest technique based on fluid recovery and genuinely measuring the mass transport of fluid does not. This review scrutinizes the nature of the evidence put forward for enterocyte secretion and reaches the conclusion that there is no evidence for it. Debilitating secretion undoubtedly does take place in severe diarrhoeal disease, but secretion from enterocytes is unlikely to be the cause.

Intracellular Autoactivation of Human Cationic Trypsinogen Mutants Causes Reduced Trypsinogen Secretion and Acinar Cell Death

Mutations in the activation peptide of human cationic trypsinogen have been found in patients with chronic pancreatitis. Previous biochemical studies demonstrated that mutations p.D19A, p.D22G, and p.K23R strongly stimulate trypsinogen autoactivation. In the present study, we characterized the cell biological effects of these mutants using human embryonic kidney 293T and AR42J rat acinar cells. We found that relative to wild-type trypsinogen, secretion of the mutants from transfected cells was markedly decreased. This apparent secretion defect was completely rescued by inhibition of autoactivation via (1) inclusion of the small molecule trypsin inhibitor benzamidine in the growth medium; or (2) cotransfection with the physiological trypsin inhibitor SPINK1; or (3) by mutation of the catalytic Ser(200) residue in trypsinogen. In contrast, extracellularly added SPINK1 or other nonpermeable proteinaceous trypsin inhibitors did not restore normal secretion of the mutants, indicating that intracellular autoactivation is responsible for the observed secretion loss. Acinar cells expressing the p.D22G mutant detached from the culture plate over time, became terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive, and exhibited elevated levels of the proapoptotic transcription factor CHOP. The observations indicate that activation peptide mutants of human cationic trypsinogen undergo autoactivation intracellularly, which leads to decreased trypsinogen secretion and eventual acinar cell death. The results thus define a novel pathological pathway for parenchymal injury in hereditary chronic pancreatitis.

A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin duringToxoplasma gondiiinfection

We have utilized a highly sensitive approach based on fluorescence resonance energy transfer (FRET) and beta-lactamase (BLA), which we adapted for the detection of Toxoplasma gondii secreted proteins. This assay revealed that the actin-binding protein toxofilin appears to be secreted into host cells during invasion. To determine the function of toxofilin during infection, we engineered a type I (RH strain) parasite with a targeted deletion of the toxofilin gene and compared the phenotypes of control and toxofilin knockout (Deltatxf) parasites in several in vitro assays, including invasion, growth, gliding motility, and egress of the Deltatxf parasites, as well as F-actin staining, phagocytosis and migration of cells infected with Deltatxf parasites or wild-type controls. Despite its apparent secretion into host cells and its ability to bind to and modulate host actin, we observed that toxofilin does not appear to play a role in these processes, under the conditions we examined, and we report these findings here.

A compartmental model describing changes in progesterone concentrations during the oestrous cycle

The objective of this study was to develop a mathematical model that accurately describes the rise and decline in plasma progesterone concentrations, and is able to define parameters describing progesterone appearance and disappearance during the bovine oestrous cycle. Daily plasma progesterone data from 27 cows were used to develop a compartmental model consisting of an appearance function and an appearance modulating function. Model outputs included an apparent appearance or secretion duration, appearance rate and an average disappearance rate (expressed as arbitrary units per day; units/d). Shape-based clustering identified three common shape-based groups (or clusters) of progesterone profiles defined as either 'peaked' profile, with the profile reaching a distinguishable peak, 'structured', with the profile exhibiting a wave-like pattern, or 'flat top', with the profile reaching a plateau. Differences in the model parameters for the three different shapes of progesterone profiles were examined: peaked (n=13), flat top (n=7) and structured (n=7). The mean duration of apparent appearance was 11.49 (SD 0.17 d) for all 27 profiles. The model estimates for total appearance of progesterone (area under the curve; ng/ml per cycle), mean appearance rate and maximum appearance rate were 69.04 ng/ml per cycle (SD 15.2 ng/ml per cycle), 3.19 ng/ml per cycle (sd 0.7 ng/ml per d) and 6.70 ng/ml (SD 1.31 ng/ml), respectively. The average disappearance rate was 1.0 units/d (SD 0.04 units/d). The apparent appearance duration was greatest (P<0.01) in the flat top profiles (12.54, SD 0.41 d) followed by the structured (11.77, SD 0.66 d) and the peaked (10.80, SD 0.30 d) profiles. Total and mean progesterone appearance, maximum progesterone appearance rate, and the progesterone disappearance rates were not different between the profiles. The model successfully simulated all components of the progesterone profile and was able to define specific parameters of different shaped progesterone profiles. A simple model able to estimate parameters describing progesterone appearance and disappearance can be used to explore the relationships between profile shapes and reproductive outcomes.

Post-harvest management of ber ( Ziziphus mauritiana Lamk) fruit rot ( Alternaria alternata Fr. Keissler) using Trichoderma species, fungicides and their combinations

Isolates of Trichoderma species from hot arid regions, fungicides and their combinations were evaluated for the management of ber fruit rot at post-harvest stage. Out of 16 isolates of Trichoderma species, six isolates checked growth of mycelia of Alternaria alternata by more than 55%. A distinct variation in the inherent level of resistance in these six isolates was recorded against 13 common fungicides. None of the Trichoderma species grew in PDA amended with carbendazim even at a very low concentration. In contrast, 100 per cent radial growth was observed in Trichoderma citrinoviride isolate T.c-CIAH224 in the presence of copper oxychloride (250 μg g −1) and mancozeb (100 μg g −1) in PDA medium. Isolate T.v-CIAH240 was antagonistic against A. alternata and tolerant to most of the fungicides tested. This isolate was highly compatible with chlorothalonil, dinocap and wettable sulphur even at 1000 μg g −1 and produced yellowish instead of normal green coloured conidia. Isolate of Trichoderma koningii – T.k-CIAH176, T. citrinoviride – T.c-CIAH224 and those of Trichoderma viride – T.v-CIAH181 and T.v-CIAH240 with inherent tolerance to some of the fungicides have shown better efficacy to suppress the fruit rot pathogen in dual cultures. However isolate T.v-CIAH240 was significantly superior in vitro in the suppression (71%) of the A. alternata through mycoparasitism and apparent secretion of secondary metabolites in the growth medium. Mycoparasitism and competition with the fruit rot pathogen were the mode of action in the majority of the isolates. The growth of A. alternata was completely inhibited in PDA amended with dinocap, propiconazole and tridemorph irrespective of the concentrations. In experiments in vivo, isolate T.v-CIAH240 was significantly effective (75% PEDC- Per cent Efficiency of Disease Control) against post-harvest infection by A. alternata followed by T.v-SBI48 (62 PEDC) and T.v-CIAH149 (44 PEDC). Among the individual fungicide treatments, dinocap and copper oxychloride (50 μg g −1) resulted in 52 PEDC. However, dinocap caused a scorching effect and a foul smell in fruits. Fruit rot control efficacy was enhanced to >70% by T.v-CIAH240 with tridemefon, thiophanate methyl, mancozeb or alcidine at 50 μg g −1 and to >80% with 100 μg g −1 of thiophanate methyl, chlorothalonil, mancozeb and alcidine in ber fruits (cv. Gola). There was a non-significant latent infection caused by isolates T.k-CIAH176, T.c-CIAH224 ( T. citrinoviride) and T.v-CIAH240. Such a latent infection was completely suppressed by low concentrations of fungicides when used in combination with T.v-CIAH240. The possible hypothesis involved in management of A. alternata in ber fruits at post-harvest stages using fungicides, Trichoderma species and their combinations is discussed.

A Case of Sebaceoma With Extensive Apocrine Differentiation

Apocrine differentiation is a rare event in sebaceoma, and only 3 cases have been reported. We report a case of sebaceoma with extensive apocrine differentiation on the scalp in a 73-year-old Japanese woman. The resected tumor was located entirely within the dermis and subcutis as a well-circumscribed, lobulated, solid, and partially cystic mass, measuring 35 mm at the largest diameter. Histopathologically, it was composed of uniform basaloid cells with clusters of sebocytes, squamous islands of ductal structures, and apocrine cells with apparent decapitation secretion. Nuclear atypia of all types of cells was inconspicuous, and mitotic figures were infrequent. We considered the lesion to be a sebaceoma with apocrine differentiation.

Prediction of human pharmacokinetics — renal metabolic and excretion clearance

The kidneys have the capability to both excrete and metabolise drugs. An understanding of mechanisms that determine these processes is required for the prediction of pharmacokinetics, exposures, doses and interactions of candidate drugs. This is particularly important for compounds predicted to have low or negligible non-renal clearance (CL). Clinically significant interactions in drug transport occur mostly in the kidneys. The main objective was to evaluate methods for prediction of excretion and metabolic renal CL (CL(R)) in humans. CL(R) is difficult to predict because of the involvement of bi-directional passive and active tubular transport, differences in uptake capacity, pH and residence time on luminal and blood sides of tubular cells, and limited knowledge about regional tubular residence time, permeability (P(e)) and metabolic capacity. Allometry provides poor predictions of excretion CL(R) because of species differences in unbound fraction, urine pH and active transport. The correlation between fraction excreted unchanged in urine (f(e)) in humans and animals is also poor, except for compounds with high passive P(e) (extensive/complete tubular reabsorption; zero/negligible f(e)) and/or high non-renal CL. Physiologically based in-vitro/in-vivo methods could potentially be useful for predicting CL(R). Filtration could easily be predicted. Prediction of tubular secretion CL requires an in-vitro transport model and establishment of an in-vitro/in-vivo relationship, and does not appear to have been attempted. The relationship between passive P(e) and tubular fraction reabsorbed (f(reabs)) for compounds with and without apparent secretion has recently been established and useful equations and limits for prediction were developed. The suggestion that reabsorption has a lipophilicity cut-off does not seem to hold. Instead, compounds with passive P(e) that is less than or equal to that of atenolol are expected to have negligible passive f(reabs). Compounds with passive P(e) that is equal to or higher than that of carbamazepine are expected to have complete f(reabs). For compounds with intermediate P(e) the relationship is irregular and f(reabs) is difficult to predict. Tubular cells are comparably impermeable (for passive diffusion), and show regional differences in enzymatic and transporter activities. This limits the usefulness of microsome data and makes microsome-based predictions of metabolic CL(R) questionable. Renal concentrations and activities of CYP450s are comparably low, suggesting that CYP450 substrates have negligible metabolic CL(R). The metabolic CL(R) of high-P(e) UDP-glucuronyltransferase substrates could contribute to the total CL.

Isorhamnetin prevent endothelial cell injuries from oxidized LDL via activation of p38MAPK

The present investigation was undertaken to determine the protective effects of isorhamnetin on endothelial cell line EA.hy926 injuries induced by oxidized low-density lipoprotein (ox-LDL) and to uncover some of the underlying mechanisms of these effects. Indices such as cell viability, lactate dehydrogenase (LDH), and nitric oxide (NO) release were measured to evaluate the protective effects of isorhamnetin. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, superoxide dismutase (SOD), superoxide and reactive oxygen species (ROS) generation were also detected to evaluate the antioxidant effects of isorhamnetin. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to confirm the expression of endothelial nitric oxide synthase (eNOS) mRNA and lectin-like ox-LDL receptor-1 mRNA. Western blotting was used to evaluate the protein expression of this receptor and eNOS, as well as p38-mitogen-activated protein kinase (p38MAPK) phosphorylation and NF-κB p65 translocation. As a result, cell viability decreased significantly ( P < 0.01) after 24 h treatment with ox-LDL, accompanied with apparent secretion disorders such as NO reduction and LDH increase. Pretreatment with isorhamnetin resulted in remarkable increase of cell viability ( P < 0.05) and modulation of secretion disorders mediated by ox-LDL in a concentration-dependent manner. Besides, ox-LDL led to upregulation of lectin-like ox-LDL receptor-1, phosphorylation of p38MAPK, translocation of NF-κB, and downregulation of the eNOS expression in endothelial cells. Isorhamnetin pretreatment inhibited the ox-LDL-induced downregulation of eNOS, upregulation of lectin-like ox-LDL receptor-1, phosphorylation of the p38MAPK and translocation of NF-κB. Moreover, isorhamnetin exhibited strong antioxidant activity, which was shown by its inhibition effects on ox-LDL-induced superoxide, ROS overproduction and significant SOD reduction. The data indicated the protective effects of isorhamnetin on endothelial cell line EA.hy926 from ox-LDL-induced cell injuries. These effects were obtained via inhibition of lectin-like ox-LDL receptor-1 upregulation, interference of ox-LDL-mediated intracellular signaling pathway (p38MAPK activation, NF-κB nuclear translocation, eNOS expression) and the antioxidant activity of isorhamnetin.

Flavonoids From Seabuckthorn Protect Endothelial Cells (EA.hy926) From Oxidized Low-density Lipoprotein Induced Injuries Via Regulation of LOX-1 and eNOS Expression

The present investigation was undertaken to determine the protective effects of flavonoids from seabuckthorn (FSBT), a traditional Chinese medicine, on endothelial cell line EA.hy926 injury induced by oxidized low-density lipoprotein (ox-LDL). Possible mechanisms were then explored. The effects of quercetin and isorhamnetin, 2 major components of FSBT, were examined as well. Indices such as cell viability, lactate dehydrogenase, nitric oxide (NO), superoxide dismutase, and superoxide were measured. Reverse transcription polymerase chain reaction, Western blot, and immunocytochemistry were employed to determine the endothelial constitutive NO synthase (eNOS) and lectinlike low-density lipoprotein receptor-1 (LOX-1) expression. Cell viability decreased significantly after 24 hours treatment with ox-LDL, accompanied with apparent secretion disorders such as NO reduction and lactate dehydrogenase increase. FSBT pretreatment could remarkably prevent both cell death and secretion disorders in a concentration-dependent manner. Besides, it was observed that ox-LDL triggered superoxide production and suppressed the superoxide dismutase activity, both of which could be prevented by FSBT pretreatment. Moreover, ox-LDL inhibited eNOS expression and increased LOX-1 expression, whereas FSBT pretreatment partly abolished these effects. Similar effects were obtained with quercetin and isorhamnetin, implying that they may contribute, at least in part, to the protective effects of FSBT. The data indicate that the protective effects of FSBT against ox-LDL induced endothelial cell injuries might derive from its antioxidant activity and its capability in modulating the expression of eNOS and LOX-1. And quercetin and isorhamnetin may contribute to these effects of FSBT.

Interaction of human osteoblasts with bioinert and bioactive ceramic substrates

The objective of this study was to compare adhesion, replication, and differentiation of human osteoblasts (OPC1 cell line) on two different ceramic substrates: a bioinert alumina (A1(2)O(3)), and a bioactive hydroxyapatite (HAp). Scanning electron microscopy (SEM) and confocal scanning microscopy were used to assess production and distribution of specific osteoblast proteins. Cells grew readily on HAp and alumina. On alumina and HAp, OPC1 cells produced distinctive cell-to-cell and cell-to-scaffold filopodia and microextensions. OPC1 cells proliferated faster on HAp than on alumina substrates. Adhesion was further assessed by observing production of the protein vinculin. On alumina, vinculin was detected throughout the cytoplasm, with some peripheral punctate regions. On HAp, OPC1 cells developed few punctate peripheral regions of vinculin; rather, the protein was strongly localized within the cytoplasm. Finally, we measured accumulation of the marker osteopontin (OPN). OPN was first detectable on day 5 and increased through day 11 of culture on alumina. OPC1 cells on HAp produced substantial amounts of OPN beginning at day 5, with apparent secretion from the cells by culture day 11. These results suggest that the function of osteoblasts is strongly affected by the properties of ceramic surfaces, and rapid attachment of osteoblasts may result in earlier differentiation on HAp than on alumina.

R-spondin, a novel gene with thrombospondin type 1 domain, was expressed in the dorsal neural tube and affected in Wnts mutants

We identified a novel gene, which encodes a 265-amino-acid sequence with a thrombospondin (TSP) type 1 motif. Unlike the other secretory proteins of the TSP family, this gene encodes no apparent secretion cleavage site, but has a putative nuclear localization signal. Northern blot analysis showed transient expression in the central nervous system (CNS) during development. In situ hybridization showed its expression in the dorsal part of the neural tube on 10 and 12 dpc, especially in the boundary region between roof plate and neuroepithelium. This expression was enhanced in the rostral part. The signals were observed in other tissues such as truncal region neighboring forelimbs and mesenchymal tissues around the nasal cavity. We named this gene R-spondin (roof plate-specific spondin). Transfection of an epitope-tagged R-spondin into COS7 and 293 cells showed its localization in nuclei and medium, suggesting that R-spondin may become secretory or nuclear protein by some processing, while most of other proteins with TSP type 1 domain are secretory proteins. The expression of R-spondin was reduced in Wnt-1/3a double knockout mouse. R-spondin might be a novel marker of the boundary between the roof plate and neuroepithelium and may contribute to the development of dorsal neural tube under the regulation of Wnts.

A calreticulin-like molecule from the human hookworm Necator americanus interacts with C1q and the cytoplasmic signalling domains of some integrins.

Calreticulin was recently identified as a hookworm (Necator americanus) allergen, implying secretion, and contact with cells of the immune system, or significant worm attrition in the tissues of the host. As human calreticulin has been shown to bind to and neutralize the haemolytic activity of the complement component C1q, and to be putatively involved in integrin-mediated intracellular signalling events in platelets, it was of interest to determine whether a calreticulin from a successful nematode parasite of humans, with known immune modulatory and antihaemostatic properties, exhibited a capacity to interfere with complement activation and to interact with integrin domains associated with cell signalling in platelets and other leucocytes. We can now report that recombinant calreticulin failed to demonstrate significant calcium binding capacity, which is a hallmark of calreticulins in general and may indicate inappropriate folding following expression in a prokaryote. Nevertheless, recombinant calreticulin retained sufficient molecular architecture to bind to, and inhibit the haemolytic capacity of, human C1q. Furthermore, recombinant calreticulin reacted in surface plasmon resonance analysis (SPR) with peptides corresponding to cytoplasmic signalling domains of the integrins alphaIIb and alpha5, in a calcium independent manner. SPR was also used to ratify the specificity of a polyclonal antibody to hookworm calreticulin, which was then used to assess the stage specificity of expression of the native molecule (in comparison with reverse transcriptase-polymerase chain reaction), to indicate its apparent secretion, and to purify native calreticulin from worm extracts by affinity chromatography. This development will allow the functional tests described above to be repeated for native calreticulin, to ascertain its role in the host-parasite relationship.

Identification and Immunogenicity of Group AStreptococcusCulture Supernatant Proteins

Extracellular proteins made by group A Streptococcus (GAS) play critical roles in the pathogenesis of human infections caused by this bacterium. Although many extracellular GAS proteins have been identified and characterized, there has been no systematic analysis of culture supernatant proteins. Proteins present in the culture supernatant of strains of serotype M1 (MGAS 5005) and M3 (MGAS 315) mutants lacking production of the major extracellular cysteine protease were separated by two-dimensional gel electrophoresis and identified by amino-terminal amino acid sequencing and interrogation of available databases, including a serotype M1 genome sequence. In the aggregate, amino-terminal amino acid sequence data for 66 protein spots were generated, 53 unique sequences were obtained, and 44 distinct proteins were identified. Sixteen of the 44 proteins had apparent secretion signal sequences and 27 proteins did not. Eight of the 16 proteins with apparent secretion signal sequences have not been previously described for GAS. Antibodies against most of the apparently secreted proteins were present in sera from mice infected subcutaneously with MGAS 5005 or MGAS 315. Humans with documented GAS infections (pharyngitis, acute rheumatic fever, and severe invasive disease) also had serum antibodies reacting with many of the apparently secreted proteins, indicating that they were synthesized in the course of GAS-human interaction. The genes encoding four of the eight previously undescribed and apparently secreted culture supernatant proteins were cloned, and the proteins were overexpressed in Escherichia coli. Western blot analysis with these recombinant proteins and sera from GAS-infected mice and humans confirmed the immunogenicity of these proteins. Taken together, the data provide new information about the molecular aspects of GAS-host interactions.

Absorption of iron in rats with experimental enteritis.

Inflammatory conditions of the gastrointestinal tract and iron-deficiency anemia are very common in humans. Acute intestinal inflammation was pathologically established in rats by intraluminal administration of acetic acid into the duodenum and the proximal jejunum. The study included two control groups of intact (untreated) rats and sham-operated (saline-treated) rats for each intestinal segment. A third group of rats received acetic acid. The acetic acid-induced inflammatory process was established histopathologically and biochemically. Two days after treatment, iron absorption was measured using ligated 10-cm loops of proximal jejunum or ligated duodenum in which 59Fe was injected intraluminally (n = 6 in each group). In another four control groups (intact and sham-operated for each intestinal segment) and two acetic acid-treated groups, serosal-luminal secretion of 59Fe was measured after intravenous injection (n = 5 in each group). 59Fe transfer from the lumens of the duodenum and jejunum to the portal system was significantly lower in those rats in whom inflammation was induced by acetic acid. There was no apparent serosal-luminal secretion of intravenously injected 59Fe in any of the studied groups. We conclude that acetic acid-induced intestinal inflammation significantly reduces iron absorption by the duodenum and the proximal jejunum.

Bicarbonate and CO2 transport across the skin of the leopard frog, Rana pipiens: effect of PGF2alpha.

The amphibian skin represents an important organ for osmoregulation and, like the mammalian kidney, maintains acid-base balance by secreting protons or base. However, the lack of a reliable and accurate method to measure the contribution of unidirectional fluxes of HCO3- ions to this mechanism has been an obstacle for the determination of the role of bicarbonate in epithelial acid-base homeostasis. Recently, one of us developed a method that allows for the reliable determination of transepithelial fluxes of bicarbonate, and this method was applied to determine unidirectional fluxes of (14)CO2 and H(14)CO3 under a variety of conditions. We report that the combined CO2 and HCO3- mucosal-to-serosal flux under 5% CO2 was 40% larger than the opposing flux, giving a net flux in the mucosal-to-serosal direction. This net flux was inhibited by acetazolamide. In CO2-free conditions, there was no detectable net flux; however, acetazolamide and PGF(2alpha) attenuated the mucosal-to-serosal flux and established an apparent secretion of HCO3-. A model is presented that depicts twelve vectors or components to the CO2 plus HCO3- fluxes in the frog skin. This model can accurately reproduce the experimental values measured from unidirectional fluxes of CO2 and HCO3- under a variety of conditions and can explain the effects of PGF(2alpha) on unidirectional 14C-labeled fluxes as a consequence of inhibition of H+ secretion to the apical bath, similar to what was previously suggested by our laboratory using a different methodological approach. The present method, utilizing radiolabeled HCO3-, may be useful as a means to evaluate the mechanism of action of hormones and drugs that may regulate acid-base homeostasis by altering proton and bicarbonate transport processes.

Tissue distribution of human gliostatin/platelet-derived endothelial cell growth factor (PD-ECGF) and its drug-induced expression

Human tissue contents of gliostatin/platelet-derived endothelial cell growth factor (PD-ECGF) and its drug-induced expression in tumor cells were currently examined by a sandwich enzyme immunoassay (EIA) system and a reverse transcription-polymerase chain reaction (RT-PCR) method. Gliostatin/PD-ECGF was found to distribute in rather ubiquitous than specific human tissues and organs, with a relatively high levels in the tissues of digestive system (esophagus and rectum), brain, spleen, bladder and lung, but not in gall bladder, aorta, muscle, fat and kidney. Most of examined human tumor cell lines showed 4- or 5-fold higher contents (21.5 ± 3.9 ng/mg protein) than normal tissue contents (4.4 ± 1.1 ng/mg protein) on the average. While gliostatin/PD-ECGF is known to lack a signal sequence, some tumor cells (A431 and MKN74) appeared to release it into the conditioned medium. Expression of gliostatin/PD-ECGF in epidermoid carcinoma cell (A431) and stomach cancer cell (MKN45) was induced by dibutyryl cyclic AMP and phorbol ester, and uniquely in MKN45 by hydrocortisone. In particular, this hydrocortisone specifically caused an increase of the apparent secretion of MKN74 without its cytotoxic effects, suggesting a possible secretion of gliostatin/PD-ECGF in the restricted but not universal cell line. Biological significance on the chemical induction of gliostatin/PD-ECGF in tumor cells and on its extracellular secretion are discussed.