Salt at concentrations relevant to meat processing enhances Shiga toxin 2 production in Escherichia coli O157:H7.

Escherichia coli (E. coli) O157:H7 remains a major food safety concern associated with meat, especially beef products. Shiga toxins (Stx) are key virulence factors produced by E. coli O157:H7 that are responsible for hemorrhagic colitis and Hemolytic Uremic Syndrome. Stx are heat stable and can be absorbed after oral ingestion. Despite the extensive study of E. coli O157:H7 survival during meat processing, little attention is paid to the production of Stx during meat processing. The objective of this study was to elucidate the effect of salt, an essential additive to processed meat, at concentrations relevant to meat processing (0%, 1%, 2%, 3%, W/V) on Stx2 production and Stx2 prophage induction by E. coli O157:H7 strains. For both E. coli O157:H7 86-24 and EDL933 strains, including 2% salt in LB broth decreased (P<0.05) E. coli O157:H7 population, but increased (P<0.05) Stx2 production (as measured relative to Log(10)CFU) compared to that of the control (1% salt). Supplementing 3% salt decreased (P<0.05) both E. coli O157:H7 number and Stx2 production. Quantitative RT-PCR indicated that stx2 mRNA expression in culture media containing 2% salt was greatly increased (P<0.05) compared to other salt concentrations. Consistent with enhanced Stx2 production and stx2 expression, the 2% salt group had highest lambdoid phage titer and stx2 prophage induction among all salt treatments. RecA is a key mediator of bacterial response to stress, which mediates prophage activation. Quantitative RT-PCR further indicated that recA mRNA expression was higher in both 2% and 3% salt than that of 0% and 1% salt treatments, indicating that stress was involved in enhanced Stx2 production. In conclusion, salt at the concentration used for meat processing enhances Stx production, a process linked to bacterial stress response and lambdoid prophage induction.

High temperature in combination with UV irradiation enhances horizontal transfer of stx2 gene from E. coli O157:H7 to non-pathogenic E. coli.

BACKGROUND: Shiga toxin (stx) genes have been transferred to numerous bacteria, one of which is E. coli O157:H7. It is a common belief that stx gene is transferred by bacteriophages, because stx genes are located on lambdoid prophages in the E. coli O157:H7 genome. Both E. coli O157:H7 and non-pathogenic E. coli are highly enriched in cattle feedlots. We hypothesized that strong UV radiation in combination with high temperature accelerates stx gene transfer into non-pathogenic E. coli in feedlots. METHODOLOGY/PRINCIPAL FINDINGS: E. coli O157:H7 EDL933 strain were subjected to different UV irradiation (0 or 0.5 kJ/m(2)) combination with different temperature (22, 28, 30, 32, and 37 °C) treatments, and the activation of lambdoid prophages was analyzed by plaque forming unit while induction of Stx2 prophages was quantified by quantitative real-time PCR. Data showed that lambdoid prophages in E. coli O157:H7, including phages carrying stx2, were activated under UV radiation, a process enhanced by elevated temperature. Consistently, western blotting analysis indicated that the production of Shiga toxin 2 was also dramatically increased by UV irradiation and high temperature. In situ colony hybridization screening indicated that these activated Stx2 prophages were capable of converting laboratory strain of E. coli K12 into new Shiga toxigenic E. coli, which were further confirmed by PCR and ELISA analysis. CONCLUSIONS/SIGNIFICANCE: These data implicate that high environmental temperature in combination with UV irradiation accelerates the spread of stx genes through enhancing Stx prophage induction and Stx phage mediated gene transfer. Cattle feedlot sludge are teemed with E. coli O157:H7 and non-pathogenic E. coli, and is frequently exposed to UV radiation via sunlight, which may contribute to the rapid spread of stx gene to non-pathogenic E. coli and diversity of shiga toxin producing E. coli.

AMP-activated protein kinase regulates beta-catenin transcription via histone deacetylase 5.

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism; it is inhibited under obese conditions and is activated by exercise and by many anti-diabetic drugs. Emerging evidence also suggests that AMPK regulates cell differentiation, but the underlying mechanisms are unclear. We hypothesized that AMPK regulates cell differentiation via altering ß-catenin expression, which involves phosphorylation of class IIa histone deacetylase 5 (HDAC5). In both C3H10T1/2 cells and mouse embryonic fibroblasts (MEFs), AMPK activity was positively correlated with ß-catenin content. Chemical inhibition of HDAC5 increased ß-catenin mRNA expression. HDAC5 overexpression reduced and HDAC5 knockdown increased H3K9 acetylation and cellular ß-catenin content. HDAC5 formed a complex with myocyte enhancer factor-2 to down-regulate ß-catenin mRNA expression. AMPK phosphorylated HDAC5, which promoted HDAC5 exportation from the nucleus; mutation of two phosphorylation sites in HDAC5, Ser-259 and -498, abolished the regulatory role of AMPK on ß-catenin expression. In conclusion, AMPK promotes ß-catenin expression through phosphorylation of HDAC5, which reduces HDAC5 interaction with the ß-catenin promoter via myocyte enhancer factor-2. Thus, the data indicate that AMPK regulates cell differentiation and development via cross-talk with the wingless and Int (Wnt)/ß-catenin signaling pathway.

Demonstration of protein absorption in the intestinal epithelium of fish and mice by laser scanning confocal microscopy.

Selective permeability for small proteins and oligopeptides occurs in the intestinal epithelium of many animal species and humans. Whole proteins are sometimes endocytosed and undergo partial hydrolysis in intestinal epithelial cells with the probable release of essential oligopeptides into the bloodstream. Increased permeability to certain proteins can cause asthma and other metabolic disorders. Permeable proteins have also been successfully used to deliver vaccines or drugs via oral consumption. Protein absorption has been inferred in many cases and demonstrated in some cases by histochemical, tracer, and analytical techniques. However, the nature and importance of protein absorption remains largely unknown. Here, we demonstrate the movement of two lumenal proteins (GFP: 26 kDa and OFP: 23 kDa) across the intestinal epithelium of fish and mice using laser scanning confocal microscopy. The results provide evidence that small proteins can be taken up intact by intestinal epithelial cells, even though large proteins are digested to single amino acids or protein fragments before they are absorbed. Our results suggest that it is possible to orally administer some small proteinous medicines for therapeutic purposes.

Proteomic identification of the silkworm (Bombyx mori L) prothoracic glands during the fifth instar stage.

Although the ecdysteroid of the silkworm had been studied for decades, the proteome of the prothoracic gland, the primary source of ecdysteroid hormones, has not been studied previously. In the present paper, we utilized a proteomic approach to investigate the fifth instar prothoracic gland during the growth and development of the silkworm, Bombyx mori L. The two-dimensional electrophoresis results showed that the majority of proteins were acidic proteins, especially concentrated in the area of 25-65 kDa, with pI values of between 4 and 7, and the difference was not distinct. When compared with Qiufeng (Japanese strain), the interspecific distinction was larger than the intraspecific distinction, and 19 particular spots, excized from the third, fifth and ninth days of p50 (Chinese strain) and Qiufeng were subjected to MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight MS) analysis. We sorted them into seven catagories: energetics and/or metabolism, storage proteins, protection, lipid metabolism, signal transduction, cell function and unknown function proteins. Of these proteins, arginine methyltransferase is discussed as playing an important role in regulating the activation of ecdysteroidogenesis via transcription or translation.

Quantification of silkworm coactivator of MBF1 mRNA by SYBR Green I real-time RT-PCR reveals tissue- and stage-specific transcription levels.

Transcriptional coactivators play a crucial role in gene transcription and expression. Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA-binding activators, such as FTZ-F1 and GCN4. Until now, very few studies have been reported in the silkworm. We selected the Bombyx mori because it is a model insect and acts as an economic animal for silk industry. In this study, we conducted the quantitative analysis of MBF1 mRNA in silkworm B. mori L. with actin (A3) as internal standard by means of SYBR Green I real-time RT-PCR method. The total RNA was extracted from the silk gland, epidermis, fat body, and midguts of the fifth instar B. mori larvae. The mRNA was reverse transcripted, and the cDNA fragments of MBF1 mRNA and actin gene were amplified by RT-PCR using specific primers. MBF1 mRNA expression in different tissues of silkworm B. mori L. was quantified using standardized SYBR Green I RT-PCR. The results suggested MBF1 gene was expressed in all investigated organs but highly expressed in the silk gland, showing its relation to biosynthesis of silk proteins.

High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system.

This novel orange fluorescent protein (OFP) emits brilliant orange fluorescent light. OFP has high fluorescence quantum yield, fast maturation rate, and stability, which imply this protein should be the most favorable biotechnological tools used to investigate the function of target gene by visualizing, monitoring, and quantifying in living cells. B. mori, silkworm has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). In this paper, we used infection technique which introduced the baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus, and demonstrated a high-level expression of the orange fluorescent protein (OFP) gene in the Bombyx mori, silkworm larvae. When recombinant rBacmid/BmNPV/OFP DNA ranging from 50-100 ng/larval was injected, a sufficient OFP expression in hemolymph was harvested. The recombinant viruses could be obtained from the hemolymph of infected larvae and stored as seed which could be used for the large-scale expression. This procedure omitted the costly and labor-consumed insect cell culture. Further investigation of OFP should provide us with more insight in unlocking the mystery of the mechanisms of autocatalytic bioluminescence and its utilization in biotechnology.

Silkworm powder containing manganese superoxide dismutase regulated the immunity and inhibited the growth of Hepatoma 22 cell in mice.

The effects of SOD contained silkworm powder on immune regulation and inhibition against Hepatoma 22 tumor cells in vivo were investigated. The activity of natural killer cell (NK) and the ConA-stimulated spleen proliferation were measured. The results found that the SOD-contained silkworm powder caused an enhancement on NK cell activity, which implied this material modulated the immune system in mice in vivo. The NK cell activities of Hepatoma 22 tumor modeled mice treated with silkworm powder including SOD were increased significantly compared to a modeled control and silkworm powder without SOD, reaching 36.18%. In addition, the ConA-stimulated spleen proliferation of SOD treated mice was higher than that of the controls. The treatment of SOD contained silkworm powder presented 40.3% of average inhibition rate to Hepatoma 22 tumor, showing stronger inhibition against tumor. There were no significant difference in body weight between modeled control and SOD silkworm powder feeding in Hepatoma 22 tumor modeled mice, suggesting the SOD silkworm powder is safety as an inhabitant to tumor. In conclusion, these findings demonstrate that administration of silkworm powder containing SOD results in activation of NK cells and immunity, suggesting the silkworm powder containing SOD plays a positive role in tumor inhibition.

Manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L enhances the NK activity and splenocyte proliferation against Sarcoma 180 tumor cells in vivo.

Natural killer cell (NK) is known as a major immune system in body through mediating cell death via several possible pathways, and one of three subpopulations of lymphocytes functioning as scavenger of tumor, virus infected cells etc. Our present results found that the SOD-contained silkworm larvae powder caused an enhancement of the effect on NK cell cytotoxicity, which implied this material modulated the immune system in mice in vivo. The NK cell activities of S180 tumor modeled mice treated with silkworm powder including SOD were enhanced significantly ranging from 30% to 48%, respectively, compare to a distilled water feeding control and silkworm powder without SOD. Meanwhile, the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control both in T cells or B cells. The average tumor weight of S180 modeled mice treated with doses of SOD-contained silkworm powder was lighter than that of water control showing the tumor inhibition rates (IR) reached to 22.51% to 37%, respectively. In conclusion, these findings demonstrate that administration of silkworm larvae powder containing SOD results in activation of NK cells and immune T-cell and B-cell, suggesting the silkworm larvae powder containing SOD play a positive role in tumor inhibition.

Expression of apalbumin1 of Apis cerana cerana in the larvae of silkworm, Bombyx mori.

Royal jelly (RJ) is a thick, milky material produced by both the hypopharyngeal and the mandibular glands of nurse honeybees. The main proteins of RJ, named apalbumins or major royal jelly proteins (MRJPs), have multiple biological functions. Apalbumin1 is the most abundant glycoprotein of RJ. In this study, Bacmid- apalbumin1 was constructed for Apis cerana cerana using the newly established Bac-to-Bac/BmNPV baculovirus expression system (BES). This procedure allowed us to obtain the recombinant A. cerana cerana ( Acc) apalbumin1 (r Accapalbumin1) from the hemolymph of silkworm larvae through the BmNPV bacmid system, 96 h postinfection. The r Accapalbumin1 was then purified by Ni-NTA spin columns and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. A 55 kDa protein with good solubility was then obtained. The peptide Ile-Phe was identified from trypsin production of r Accapalbumin1. Such a peptide has been reported to have an antihypertensive ability. Our results have therefore potential applications in biomedical research and open new perspectives for the study of apalbumins.

Improvement of recombinant baculovirus infection efficiency to express manganese superoxide dismutase in silkworm larvae through dual promoters of Pph and Pp10.

The silkworm, Bombyx mori, has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). There are several problems which will probably be the bottleneck for practical and industrial utilization of silkworm bioreactor. Traditionally, the recombinant virus should infect the larvae through individual dorsal injection by a syringe. This is a time- and labor-consuming procedure. This drawback has become a bottleneck for practical and industrial utilization of baculovirus expression system in the silkworm bioreactor. In this paper, we constructed a dual expression baculovirus to express the renovated polyhedron and target manganese superoxide dismutase (SOD) gene under P10 and polyhedron promoters, respectively, through oral infection. The results showed that the direct injection of recombinant rBacmid/BmNPV/SOD DNA with cellfectin reagent infected the silkworm larvae partially. When next batches of larvae were fed orally with hemolymph, which was collected from first batch of injected and infected larvae, the obvious symptom of infection was found and high target SOD was expressed. These results imply it is feasible to express target genes through combination of recombinant bacmid DNA injection and oral feeding by a dual expression bacmid baculovirus.

Effects of silkworm larvae powder containing manganese superoxide dismutase on immune activity of mice.

To study the function of silkworm larvae powder containing superoxide dismutase and potential practical development, we investigated the safety assessment and effects on immune activity of mice such as the growth of immunity-related organs, delayed-type hypersensitivity (DTH) and charcoal particle clearance ability. The mean body weights in treated mice were significantly heavier than that of control, meanwhile, the ratio of splenocytes/body weight and the thoracic gland/body weight in treated mice was significantly enhanced after 30 days treated with silkworm larvae powder containing manganese superoxide dismutase. The treated mice resulted in a profound activation of the DTH and charcoal particle clearance, and indicated the treated mice have stronger phagocytic activity to exogenous materials. Our data also indicated the feeding treatment was safe with 360 folds of recommended human dosage in acute toxic test. In long-term test, there were no effects of silkworm larvae powder containing SOD on treated mice's growth and inside organs as long as 90 days. Further the electronic microscope investigation showed the intestine, liver, splenocyte and stomach in mice were no obvious changes both in organs and sub-organs such as nucleus, endoplasmic reticulum, mitochondrion, Golgi and peroxisomes after treated for as long as 90 days.

Anti-oxidation and immune responses in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L.

With manganese superoxide dismutase expressed in silkworm larvae, Bomby mori L, we investigate the effects of silkworm larvae powder containing SOD on the antioxidation and the immune system of mouse. The contents of MDA both in mice plasma or liver organ treated with silkworm larvae powder containing manganese superoxide dismutase were reduced compare to control. The superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities both in plasma or liver organ of the treated mice were significantly higher than that of both control and bromobenzene treated mice (group-BM), suggesting the silkworm larvae powder containing SOD play a positive role in anti-oxidation in mice. This experiment was also designed to investigate the effects of silkworm larvae powder containing SOD on the immune system of mouse, focused on hemolysin response, hemagglutination against SRBC and the activity of natural killer (NK) cells. All treated mice showed significant increase in hemolysin response to SRBC and demonstrated an activation of NK cell function by the SOD-contained silkworm larvae powder, which suggest a promotion in humoral immunity. The results suggested the SOD expressed in silkworm maybe have potential application in medicine.

Immunity promotion and proteomic identification in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae.

With manganese superoxide dismutase (SOD) expressed in silkworm larvae, Bomby mori L, we investigated the effects of silkworm larvae powder containing SOD on the immune system of mouse and employed a proteomics approach to examine this phenomenon. Our data on the effects of continuous treatment with SOD-containing silkworm larvae powder showed that the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control. The results of PFC assay also revealed that antibody production was higher in all three treated groups than controlled mice. We investigated the phagocytosis of mouse macrophages. The SOD treatment led to a dose-dependent increase of phagocytic activity. We identified six proteins that related to immunity of mice. The data showed all these six matched proteins related immunity presented the increase of expression level in plasma of mouse administrated with silkworm powder including SOD compared to that of control. These findings demonstrate that administration of silkworm larvae powder containing SOD results in enhancement of immunity activities in the mouse. The results also suggested that the SOD expressed in silkworm maybe have potential application in medicine.

Proteomic analysis of the silkworm (Bombyx mori L.) hemolymph during developmental stage.

We utilized the proteomic approach to investigate the proteome of the fifth instar hemolymph during growth and development, and to improve the understanding of this important bioprocess and gene expression situation. A total of 25 microL of hemolymph was used for 2D analysis, and the separated proteins were visualized by silver staining and analyzed using the ImageMaster 2D software. The report showed as many as 241 of protein spots were expressed in the beginning of the fifth instar. Among them, most were concentrated in pI 3.5-6.5, which reached 76% of the total protein spots. As for the protein molecular sizes, 182 protein spots concentrated between 35 and 90 kDa, which comes to 75% of the total spots. When the larvae grow to the seventh day (total fifth instar duration was 9 days), 298 protein spots were visualized through 2D electrophoresis. Fifty-seven spots were newly expressed compared to the image of the first day in fifth instar. The results implied that these proteins are related to biosynthesis of silk protein and metamorphosis preparation from larva to pupa. In total, 19 protein spots including 6 special spots expressed in seventh day were analyzed through MALDI-TOF-MS. The relations between proteins and growth and development of silkworm were discussed.