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The glycosylation profile of a recombinant protein is important because glycan moieties can play a significant role in the biological properties of the glycoprotein. Here we determined the site-specific N-glycosylation profile of human lactoferrin (hLF) and recombinant human lactoferrin (rhLF) expressed in the milk of transgenic cloned cattle. We used combined approaches of monosaccharide composition analysis, lectin blot, glycan permethylation and sequential exoglycosidase digestion and analyzed samples using high-performance ion chromatography and mass spectrometry (MS). N-glycans from hLF are comprised entirely of highly branched, highly sialylated and highly fucosylated complex-type structures, and many contain Lewis(x) epitopes. Six of these structures are reported here for the first time. However, N-glycans from rhLF are of the high mannose-, hybrid- and complex-type structures, with less N-acetylneuraminic acid and fucose. Some contain a terminal N-acetylgalactosamine-N-acetylglucosamine (LacdiNAc) disaccharide sequence. Monosaccharide composition analysis of rhLF revealed small amounts of N-glycolylneuraminic acid, which were not detected by MS. hLF and rhLF appear to be glycosylated at the same two sites: Asn138 and Asn479. The third putative glycosylation site, at Asn624, is unglycosylated in both hLF and rhLF. The relative abundance of each N-glycan at each site was also determined. The different N-glycosylation profile of rhLF when compared with that of hLF is in consistent with the widely held view that glycosylation is species- and tissue/cell-specific. These data provide an important foundation for further studies of glycan structure/function relationships for hLF and rhLF and help to better understand the glycosylation mechanism in bovine mammary epithelial cells.

The effects of almond consumption on DNA damage and oxidative stress among cigarette smokers were studied. Thirty healthy adult male regular smokers were randomly divided into three groups, 10 subjects per group. Group A (control group) did not receive any almonds. Subjects in Groups B and C received 3 oz and 6 oz (84 g and 168 g) of almonds each day respectively for 4 wk. Two known biomarkers for DNA damage, urinary 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and single strand DNA breaks of peripheral blood lymphocytes, were measured by enzyme-linked immunosorbent assay and comet assay, respectively. In addition, plasma malondialdehyde (MDA) level, superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activities were measured as biomarkers for oxidative stress. The results showed lower levels of urinary 8-OH-dG and single strand DNA breaks in the two almond-treated groups as compared with the control group. Furthermore, MDA levels in the almond-treated groups were lower than the controls. However, no significant effects of almonds on SOD and GSH-Px activities were found. In conclusion, results from this pilot study indicate that almond consumption has preventive effects on oxidative stress and DNA damage caused by smoking. A larger, randomized, placebo-controlled clinical trial on almonds will be initiated in the near future.

BackgroundThe dysbiosis of intestinal microbiota has been established in Crohn's disease (CD), but the molecular characterization of this dysbiosis in Chinese subjects with CD remains unclear. This study aims to investigate the predominant bacterial composition of the faecal and mucosal-associated microbiota in Chinese CD patients using culture-independent techniques.Methods/Principal FindingsEighteen patients with CD and 9 healthy controls were included in this study. The faeces and the intestinal mucosal tissues from the ulcerated and nonulcerated sites were subjected to bacterial community fingerprinting using denaturing gradient gel electrophoresis (DGGE). The predominant bacterial composition in the faeces and mucosa was determined with DNA sequencing and BLAST. We showed that the bacterial diversity in the faeces of CD patients was reduced compared with that in healthy controls (p<0.01). The faecal bacterial dysbiosis of the patients was characterized by an elevated abundance of γ-Proteobacteria (especially Escherichia coli and Shigella flexneri) and a reduced proportion of Bacteroidetes and Firmicutes. Five bacterial species defined the microbiota imbalance of the ulcerated mucosa in CD, including an increase in Escherichia coli, a decrease in Faecalibacterium prausnitzii, Lactobacillus coleohominis, Bacteroides sp and Streptococcus gallolyticus in the bacterial community as compared with the nonulcerated (p<0.01).Conclusions/SignificanceThis is the first description of intestinal microbiota dysbiosis in Chinese CD patients. These results allow a better understanding of the faecal and mucosal microbiota in CD, showing a predominance of some opportunistic pathogenic bacteria and a decrease in beneficial bacterial species. The findings may provide novel insights into the pathogenesis of CD in Chinese population.

Adhesion of lactobacilli to the host gastrointestinal (GI) tract is considered an important factor in health-promoting effects. However, studies addressing the molecular mechanisms of the adhesion of lactobacilli to the host GI tract have not yet been performed. The aim of this work was to identify Lactobacillus reuteri surface molecules mediating adhesion to intestinal epithelial cells and mucins. Nine strains of lactobacilli were tested for their ability to adhere to human enterocyte-like HT-29 cells. The cell surface proteins involved in the adhesion of Lactobacillus to HT-29 cells and gastric mucin were extracted. The active fractions were detected by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and Western blotting with horseradish peroxidase-labeled mucin and NHS-Biotin-labeled HT-29 cells. Furthermore, tandem mass spectrometry analysis was performed to identify the surface protein that participates in adhesion. It was shown that the ability of lactobacilli to adhere to HT-29 cells in vitro varied considerably among different strains. The most adhesive strain was the chicken intestinal tract isolate Lactobacillus reuteri JCM1081 (495.07 ± 80.03 bacterial cells/100 HT-29 cells). The adhesion of L. reuteri JCM1081 to HT-29 cells appeared to be mediated by a cell surface protein, with an approximate molecular mass of 29 kDa. The peptides generated from the 29-kDa protein significantly matched the Lr0793 protein sequence of L. reuteri strain ATCC55730 (*71.1% identity) and displayed significant sequence similarity to the putative ATP-binding cassette transporter protein CnBP.

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Brevibacillus laterosporus G4, which was isolated from soil sample, kills free-living nematodes (Panagrellus redivius) and plant-parasite nematodes (Bursaphelenchus xylophilus) and degrades their cuticle in previous bioassay. Our works for B. laterosporus G4 had demonstrated that an extracellular alkaline protease BLG4 played a key role as a pathogenic factor in infection against nematode. In this study, the nematicidal activity of BLG4 was further verified by an in vitro assay with purified recombinant BLG4. The encoding gene of BLG4 was cloned and showed high degree of homology with the subtilisin subclass of serine protease gene and another reported cuticle-degrading protease gene from nematophagous bacterium Bacillus sp. B16. Deletion of BLG4 by homologous recombinant had a significant effect on the pathogenicity of B. laterosporus. In infection assays the BLG4-deficient strain (BLG4-6) lost about 50% of its nematocidal activity and in toxicity tests the mortality rate of nematodes decreased with approximately 56% in comparison to wild-type strain. This is the first report analyzing the function of a subtilisin enzyme involved in bacterium against nematode at the molecular level, and it is possible to use B. laterosporus as a model to study host-parasite interaction and to gain detailed knowledge of the infection process.

Lactoferrin (LF) plays an important role in the body's immune system. However, the immunomodulatory effects of supplementation transgenic cow's milk containing recombinant human LF (rhLF) on the systemic and intestinal immune systems in infants remain unclear. Our laboratory has used genetic engineer to produce transgenic cow secreted rhLF. To assess the immune responses we took piglets as an animal model for infants. Eighteen piglets at 7 days of age were fed ordinary milk, 1:1 mix of ordinary and rhLF milk, or rhLF milk (LFM) for 30 days. The incidence of diarrhea in piglets in natural condition was observed. The protein abundances of immunoglobulin (Ig)G, IgA, IgM, IgE, histamine, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12 interferon, tumor necrosis factor in the plasma, spleen or intestine were measured by enzyme-linked immunosorbent assay. Intestinal structure was assessed by hematoxylin and eosin. The mRNA levels of immune and allergy-related genes were measured by quantitative reverse transcription-polymerase chain reaction. The results showed that LFM-fed significantly reduced incidence of diarrhea, enhanced humoral immunity, T helper (Th) 1, and Th2 cell responses, improved the structure of the intestinal mucosal and did not induce food allergy. LFM increased mRNA levels of toll-like receptor 2 and nuclear factor-κB p65 and decreased that of FCεRI β. In conclusion, rhLF-enriched formula could improve systematic and intestinal immune responses and did not elicit food allergies in neonatal piglets.