Flagellar proteins and type III-exported virulence factors are the predominant proteins secreted into the culture media of Salmonella typhimurium

We analysed all major proteins secreted into culture media from Salmonella typhimurium. Proteins in culture supernatants were collected by trichloroacetic acid precipitation, separated in SDS-polyacrylamide gels and analysed by amino acid sequencing. Wild-type strain SJW1103 cells typically gave rise to nine bands in SDS gels: 89, 67, 58, 52, 50, 42, 40, 35 and (sometimes) 28 kDa. A search of the sequences in the available databases revealed that they were either flagellar proteins or virulence factors. Six of them were flagella specific: FlgK or HAP1 (58 kDa), FliC or flagellin (52 kDa), FliD or HAP2 (50 kDa), FlgE or hook protein (42 kDa), FlgL or HAP3 (35 kDa) and FlgD or hook-cap protein (28 kDa). The other four bands were specific for virulence factors: SipA (89 kDa), SipB (67 kDa), SipC (42 kDa) and InvJ (40 kDa). The 42 kDa band was a mixture of FlgE and SipC. We also analysed secreted proteins from more than 30 flagellar mutants, and they were categorized into four groups according to their band patterns: wild type, mot type, polyhook type and master gene type. Virulence factors were constantly secreted at a higher level in all flagellar mutants except a deltamot (motAB deletion) mutant, in which the amounts were greatly reduced. A new morphological pathway of flagellar biogenesis including protein secretion is presented.     

Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted proteins during fed-batch culture

A gratuitous induction system in the yeast Kluyveromyces lactis was evaluated for the expression of intracellular and extracellular products during fed-batch culture. The Escherichia coli lacZ gene (beta-galactosidase; intracellular) and MFalpha1 leader-BPTI cassette (bovine pancreatic trypsin inhibitor; extracellular) were placed under the control of the inducible K. lactis LAC4 promotor, inserted into partial-pKD1 plasmids, and transformed into a ga1-209 K. lactis strain. To obtain a high level of production, culture conditions for growth and expression were initially evaluated in tube cultures. A selective medium containing 5 g/L glucose (as carbon source) and 0.5 g/L galactose (as inducer) demonstrated the maximum activity of both beta-galactosidase and secreted BPTI. This level of expression had no significant effect on the growth of the recombinant cells; growth rate dropped by approximately 11%, whereas final biomass concentrations remained the same. In shake-flask culture, biomass concentration, beta-galactosidase activity, and BPTI secreted activity were 4 g/L, 7664 U/g dry cell, and 0.32 mg/L, respectively. Fed-batch culture (with a high glucose concentration and a low galactose [inducer] concentration feed) resulted in a 6.5-fold increase in biomass, a 23-fold increase in beta-galactosidase activity, and a 3-fold increase in BPTI secreted activity. The results demonstrate the success of gratuitous induction during high-cell-density fed-batch culture of K. lactis. A very low concentration of galactose feed was sufficient for a high production level.     

Collagen-binding proteins secreted by type II pneumocytes in culture

The alveolar epithelial basement membrane is believed to play important roles in lung development, in maintaining normal alveolar architecture, and in guiding repair following lung injury. However, little is known about the formation of this structure, or of the mechanisms which mediate interactions between the epithelium and specific matrix macromolecules. Since type IV collagen is a major structural component of basement membranes, we investigated the production of type IV collagen-binding proteins by primary cultures of rat lung type II pneumocytes. Cultures were labeled for up to 24 h with 3H-labeled amino acids or [3H]mannose. Soluble collagen-binding proteins which accumulated in the culture medium were isolated by chromatography on collagen-Sepharose and examined by SDS-polyacrylamide gel electrophoresis. The major type IV collagen-binding protein (CBP1) was identified as fibronectin. We also identified a novel disulfide-bonded collagen-binding glycoprotein (CBP2; Mr = 45,000, reduced). This protein was not recognized by polyclonal antibodies to fibronectin, and showed no detectable binding to denatured type I collagen. The protein was resolved from fibronectin and partially purified by sequential chromatography on gelatin and type IV collagen-Sepharose. We suggest that type II pneumocyte-derived collagen-binding proteins contribute to the formation of the epithelial basement membrane and/or mediate the attachment of these cells to collagenous components of the extracellular matrix.     

Liposome dehydration on nitrocellulose and its application in a biotin immunoassay.

The feasibility of utilizing dehydrated liposomes in the development of a simple immunoassay device for point-of-care diagnostics or field assays was demonstrated. The recovery of liposomes after a cycle of dehydration and rehydration was studied using biotin-tagged, dye-loaded liposomes with antibiotin antibodies immobilized in a defined zone on nitrocellulose strips. Liposomes were vacuum-dehydrated on the strip at a location below the antibiotin zone. The strip was placed in a tube containing a carrier solution and capillary action brought the solution to the dehydrated liposomes, rehydrated them, and caused them to migrate to the antibody zone where intact liposomes were captured and measured optically. High concentrations of either trehalose or sucrose external to the liposomes and both polyvinylpyrrolidone and gelatin in the membrane blocking reagent were essential for preservation of the dehydrated/rehydrated liposomes on nitrocellulose. Between 70 and 80% of the liposomes were recovered on the nitrocellulose strips after a cycle of dehydration and rehydration. The dehydrated liposomes on the strips were stable for at least 1 year when stored in vacuum-sealed plastic bags at 4 degrees C. The technique was successfully applied to the development of a rapid one-step strip immunoassay for biotin.     

Isolation and characterization of hydroxyproline-containing proteins secreted by a murine carcinoma cell culture

A collagenous protein was isolated from a murine carcinoma cell culture, which has been shown to synthesize basement membrane. The molecular weight of this protein was estimated to be 155 000. It eluted from carboxymethyl-cellulose in the region near the alpha 1 and beta 11 components of calf skin collagen. 63--69% of the peptide-bound prolines were hydroxylated, and the 4-/3-hydroxyproline ratios ranged from 12 : 1 to 14 : 1. About 95% of the hydroxylysines in the peptide were glycosylated, and almost all of them were in the glucosylgalactosyl dissacharide form. Judging from the posttranslational characteristics, this collagenous protein is probably of basement membrane type.     

Use of nitrocellulose membranes as a scaffold in cell culture

Nitrocellulose membranes, one of the most important and oldest cellulose derivatives, are commonly used for nucleic acid and protein detection in research and diagnostic applications. However, a limited number of studies have explored whether they can act as scaffolds for cell growth. In this study, we investigated this polymeric material for its ability to support the growth of human cells. Eight established cell lines were examined for adherence, growth, spread, and survival on nitrocellulose membranes by optical microscopy after hematoxylin and eosin and/or immunocytochemical staining and by scanning electron microscopy. Apoptosis and leakage of lactate dehydrogenase (LDH) were also assessed. All cells readily adhered to and spread on the surface of nitrocellulose membranes as well as coverslips, and the cells maintained the expression of digestive system-specific genes. No significant change was detected in apoptosis or leakage of LDH from cells grown on nitrocellulose membranes. These results suggested that nitrocellulose membranes have a suitable cytocompatibility towards human cells and that they might be used for tissue-engineering scaffolds. Moreover, we demonstrate an additional and underused property of nitrocellulose of specific relevance to microscopic imaging, as it can be rendered virtually transparent, thus the cells growing on such membranes can be observed directly under an optical microscope after staining.     

Antifreeze proteins are secreted by winter rye cells in suspension culture

During cold-acclimation, winter rye ( Secale cereale L) leaves secrete antifreeze proteins (AFPs) into the apoplast. The AFPs bind to ice and modify its growth, which is easily observed in vitro . However, it is not yet known whether in planta AFPs interact with ice or whether they exert cryoprotective effects. These experiments are difficult to conduct with intact plants, so the aim of this work was to determine whether AFPs are produced in response to cold temperature in cell culture and to examine their function by using suspension cells. We showed that suspension cells secreted three of the six known winter rye AFPs into the culture medium during acclimation at 4°C. These AFPs were not present in washed suspension cells, thus indicating that they are not firmly bound to the cell walls. In order to examine the function of extracellular AFPs, non-acclimated (NA) winter rye suspension cells and protoplasts isolated from NA winter rye leaves were then frozen and thawed in the presence of AFPs extracted from cold-acclimated winter rye leaves. The AFPs had no effect on the survival of NA protoplasts after freezing; however, they lowered the lethal temperature at which 50% of the cells are killed by freezing (LT₅₀) of NA suspension cells by 2.5°C. We conclude that low above-zero temperatures induce winter rye suspension cells to secrete AFPs free in solution where they can protect intact suspension cells, but not protoplasts, from freezing injury, presumably by interacting with extracellular ice.     

Differences in proteins secreted by human fibroblasts and muscle cells in culture

Cell cultures of human skin fibroblasts, myoblasts, and fused muscle cells were grown in the presence of [¹⁴C]leucine or a mixture of [¹⁴C]amino acids. The proteins synthesised and secreted or leaked into the culture medium during radio-labelling were separated by one and two-dimensional PAGE and detected by fluorography. Four major bands of M_r 54 kD, 52 kD, 51 kD, and 49 kD were present at greatly increased concentration in fibroblast media. These fibroblast-specific polypeptides can be readily detected in myoblast/fibroblast cocultures with fibroblast content as low as 5%.     

An enzyme immunoassay for the quantitation of dihydropteridine reductase

A competitive, enzyme-linked immunosorbent assay for the quantitative determination of dihydropteridine reductase (DHPR) is described. This highly sensitive method can determine the content of DHPR protein in tissue preparations independently of the enzymatic activity of the protein molecule. The method involves initial incubation of samples containing soluble enzyme in microtiter plates coated with purified goat antibodies to rat DHPR and further incubation with DHPR conjugated to alkaline phosphatase. The assay is used to study the ontogeny of DHPR in rat liver.     

A method for the identification of proteins secreted by lactic acid bacteria grown in complex media

Lactic acid bacteria (LAB) are known for their special nutritional requirements, being usually cultured in complex media to achieve optimal growth. In this paper, a protocol based on trichloroacetic acid precipitation of peptides and proteins is presented. The method has been tested on four probiotic LAB strains grown in De Man Rogosa Sharpe (MRS) broth, a complex medium that is often used for the culture of such bacteria. This protocol allowed the detection of 19 proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 10 of them being successfully identified by tandem MS. Thereafter, the 10 were found to be secreted or surface associated by bioinformatic means. In conclusion, this work supplies a method for the identification of proteins secreted by LAB, allowing discrimination between the proteins present in the MRS and those produced by probiotic LAB.     

Noninvasive characterization of metabolites secreted in culture media by bovine embryos during in vitro production

Introduction

Secreted molecules could be correlated with the potential of embryonic development. The development of new technologies, such as mass spectrometry (MS), has enabled analyzes in culture medium to favor the determination of embryos viability in order to improve embryo selection.

Objectives

To perform a non-invasive characterization of the secretome of in vitro produced embryos with different kinetics of cleavage and in different stages of development to obtain specific patterns based on embryonic phenotype through MALDI–TOF–MS.

Methods

Bovine embryos were produced in vitro by standard protocols. The zygotes were transferred to individual culture medium and divided into two groups: Fast [4 cells-22 hours past the beginning of culture (hpc)] and Slow (2 cells-22 hpc). Culture media drops were collected at 22, 96 and 168 hpc. Analysis of embryonic secretome was made by MALDI–TOF–MS after extractions of the metabolites. Spectra were acquired in positive ionization mode. Univariate (Fold-change) and multivariate (Partial Least Squares Discriminants Analysis) analyses were performed by the online software Metaboanalyst.

Results

It was demonstrated that embryos with different kinetics have different spectrometric profiles during embryonic development. Moreover, secreted molecules in each developmental stage are differentially represented in embryos with different kinetics, and are related to specific pathways such as lipid and amino acids metabolism and cell proliferation.

Conclusion

We propose that the analysis of culture media by MALDI–TOF–MS can be used for qualitative characterization of bovine embryos, allowing the identification of key molecules during in vitro culture.

     

A streamlined method for the isolation and quantitation of nanomole levels of exported polyamines in cell culture media

A number of years ago, our laboratory published a method for the isolation of small amounts of polyamines from cell culture media using the ion-exchange resin Bio-Rex 70. We have used this technique extensively to study the export of putrescine and cadaverine from cultured mammalian cells. Unfortunately, this method was highly inefficient in isolating the polyamines spermidine and spermine and was incapable of recovering the acetylated polyamine N(1)-acetylspermidine. In response to these shortcomings, we modified our previous protocol to quantitatively isolate the polyamines N(1)-acetylspermidine, putrescine, cadaverine, N(1)-acetylspermine, spermidine, and spermine. The new method, which is much faster to perform and more efficient than the one previously described, employs the use of disposable minicolumns and a single resin washing step using a weak solution of sodium carbonate at pH 9.3. This new protocol also eliminates the column elution step in favor of directly derivatizing the polyamines with dansyl chloride on the ion-exchange resin. High-performance liquid chromatography analysis of the dansylated polyamines isolated by this procedure showed that 75% of N(1)-acetylspermidine and nearly 100% of the other polyamines present in nanomolar levels were recovered from small amounts of cell culture medium. This new protocol is a valuable new tool for the study of the intracellular/extracellular dynamics of polyamine pools in cultured cells. [A detailed laboratory protocol for this procedure (containing all of the information in this paper but in a condensed form) can be requested by e-mailing the authors.]     

Iodination of proteins on nitrocellulose blotting paper

A procedure is described for radioiodination to a high specific activity of proteins immobilized on nitrocellulose membranes. After radioiodination, the proteins can be removed from the nitrocellulose to perform such structural analyses as proteolytic mapping. This method allows the detection of small amounts of protein recognized by immunoblotting and may be used to compare the structure of immunologically related proteins.     

Use of a monoclonal antibody for quantitation of rabies vaccine glycoprotein by enzyme immunoassay

The use of a monoclonal antibody specific for the envelope glycoprotein of rabies virus has been used in a direct enzyme immunoassay to quantify the glycoprotein content of rabies vaccines produced in two kinds of cell culture. The results of this direct enzyme immunoassay are well correlated with the in vivo NIH potency test. This test may be a useful tool to rapidly and accurately control the antigenic value of a vaccine. It could also be used for the "in process' control of vaccine production before deciding whether a vaccine batch may reasonably be subjected to the NIH potency test.     

Matricellular proteins in cancer: a focus on secreted Frizzled-related proteins

Tumours are complex entities, wherein cancer cells interact with myriad soluble, insoluble and cell associated factors. These microenvironmental mediators regulate tumour growth, progression and metastasis, and are produced by cancer cells and by stromal components such as fibroblast, adipocytes and immune cells. Through their ability to bind to extracellular matrix proteins, cell surface receptors and growth factors, matricellular proteins enable a dynamic reciprocity between cancer cells and their microenvironment. Hence, matricellular proteins play a critical role in tumour progression by regulating where and when cancer cells are exposed to key growth factors and regulatory proteins. Recent studies suggest that, in addition to altering Wingless (Wnt) signalling, certain members of the Secreted Frizzled Related Protein (sFRP) family are matricellular in nature. In this review, we outline the importance of matricellular proteins in cancer, and discuss how sFRPs may function to both inhibit and promote cancer progression in a context-dependent manner. By considering the matricellular functionality of sFRPs, we may better understand their apparently paradoxical roles in cancers.     

Development of a one-step enzyme immunoassay for the quantitation of gibberellin A3 in barley seeds

The development of a sensitive and specific enzyme immunoassay for GA₃ is reported. This method was based on the use of peroxidase labelled GA₃ and immobilized antibodies. In order to obtain a rapid immunoassay, several steps of purification were analyzed to show their necessity. Barley seed extracts were assayed at different steps of purification to exhibit the effect of extract components on the assay. It was demonstrated that HPLC had to be performed when a selective quantitation of GA₃ was required. This assay allowed GA₃ to be measured with reproducibility as its unmethylated form and the quantitation of GA₃ in barley seeds with this enzyme immunoassay was correlated to a GC-MS method.Abbreviations GA₃ gibberellin A₃ - EIA enzyme immunoassay - DMF dimethylformamide - TEA tri(n)ethylamine - BSA bovine serum albumin - OVA ovalbumine - ECF ethylchloroformate - PB phosphate buffer     

A sensitive method for staining proteins transferred to nitrocellulose sheets

A simple physical method to determine the monomer concentration of detergents below as well as above the critical micelle concentration based on the bubble-pressure measurement is described. Aggregated surfactant molecules (micelles) and phospholipid vesicles if present in the sample will not disturb the measurements. Three applications of the method relevant to the preparation of liposomes are shown: (i) measurements of critical micelle concentrations, (ii) evaluation of the affinity constant of the interaction of detergents with liposomal membranes, and (iii) monitoring of residual detergent in liposome preparations during dialysis or after gel chromatography of mixed micelle-derived liposomes. It was found that the efficiency of detergents to produce liposomes during their removal depends on their critical micelle concentrations as well as on their affinity to liposomal membranes.