IgA response of BALB/c mice to orally administered Salmonella typhimurium flagellin‐displaying T2 bacteriophages

Salmonella typhimurium antigens were displayed on the capsid of a T2 bacteriophage to explore the potential of phage display for an oral vaccine. Segments of the flagellin proteins FliC (H1 antigen) and FljB (H2) were fused to the N‐terminal of T2 phage SOC to give two recombinant phages, T2FliCm and T2FljBm. Over 14 days, 19 BALB/c mice were orally administered twice, either with purified recombinant FliCm and FljBm protein, or T2FliCm and T2FljBm with or without host Escherichia coli. Feces were sampled over 10 weeks and examined for phage by plaque assay and for the presence of mucosal IgA by ELISA. Relatively few phages were detected relative to the amount administered (up to 8.21 × 10³ PFU/g faeces) and none were detected five days after initial administration. The administration of a large number of phages appeared to cause no clinical symptoms. IgA concentration in feces peaked around four weeks after the second administration and subsided after eight weeks. The highest relative titers were observed in the protein group (0.37% for anti‐FliCm and 0.22% for anti‐FljBm) and the mouse group which received no E. coli (0.33% and 0.35%) despite the theoretical amount of protein contained in a phage dose being at least 80–465 times lower than the protein dose administered. The possibility that the immuno‐stimulatory properties of the phage create an adjuvant effect to enhance the immunogenic properties of the displayed proteins is discussed. We conclude that phage may be valuable as a vector for oral vaccines. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Differential effects of platelet rich plasma and washed platelets on the proliferation of mouse MSC cells

Multipotent mesenchymal stem cell (MSC) therapies are being tested clinically for a variety of disorders. However, despite the remarkable clinical advancements in this field, most applications still use traditional culture media containing fetal bovine serum. Platelet-rich plasma (PRP) appears as a novel application for tissue engineering and its effect on bone healing is thought to be mainly dependent on the proliferation promoting function, with the molecular mechanisms largely unknown. In this study, mouse osteogenic progenitor mesenchymal stem cells (MSCs) were cultured in PRP or washed platelet (WPLT)-treated wells or in untreated wells, and analyzed on cycloxygenase 2 (COX2) expression (qRT-PCR), cell growth (MTT assay) and cell differentiation (alkaline phosphatase activity). The results showed that PRP and WPLT stimulated cell growth similarly in the first 6 days, together with the steady induction of COX2 and PGE2. 10 μmol/l celecoxib (an inhibitor of COX2) significantly inhibited the pro-proliferation effects. Interestingly, WPLT had stronger effects than PRP in proliferation at the later time points (6–9 days). ALP activity assay and collagen 1a expression revealed PRP had a mild but statistically significant pro-differentiation effect, while no obvious effects observed in WLPT group. In summary, PRP stimulates initial growth of MSCs in a COX2 partially dependent manner and the less obvious osteogenic differentiation promoting effects of WPLT strongly indicates WPLT rather than the PRP should be the optional choice for expanding MSCs in vitro for clinical use.     

Avian Podocytes,Which Lack Nephrin,Use Adherens Junction Proteins at Intercellular Junctions

Nephrin, a major intercellular junction (ICJ) molecule of mammalian podocytes in the renal glomerulus, is absent in the avian genome. We hypothesized that birds use ICJ molecules other than nephrin in their podocytes. Therefore, in the present study, we examined the possible involvement of adherens junction (AJ) proteins in the ICJs of avian podocytes. We found the AJ proteins N-cadherin and α- and β-catenins in podocytes of quail and chickens but not in those of rats, pigs or humans. The AJ proteins were prominent in avian glomerulus-rich fractions in immunoblot analyses, and in immunofluorescence microscopy analyses, they were localized along glomerular capillary walls appearing in at least two staining patterns: weakly diffuse and distinctly granular. Immunoelectron microscopy demonstrated that the significant accumulation of immunogold particles for the AJ proteins were especially evident in avian slit diaphragms and AJs. Furthermore, N-cadherin was found to be expressed in all nephron cells in the early developmental stage but became confined to podocytes during maturation. These results indicate that avian slit diaphragms clearly express AJ proteins as compared with that in the mammal—where AJ proteins are suppressed to an extremely low level—and that avian podocytes are interconnected by AJs per se in addition to slit diaphragms.     

Noninvasive sampling of DNA from larval exuvia in diving beetles of the genera Cybister and Dytiscus

Nonlethal DNA sampling is a highly recommendable method in molecular genetic studies of protected and endangered species. To develop a demonstrably nonlethal method of obtaining DNA from larvae of endangered diving beetles (Cybister brevis, C. lewisianus, C. limbatus, C. rugosus, Dytiscus sharpi sharpi and D. sharpi validus), we obtained the larval exuvia (molted skin) of these endangered diving beetles under laboratory conditions. A single exuvia 24 h after molting was sufficient to allow polymerase chain reaction (PCR) detection of a mitochondrial DNA gene, cytochrome‐c oxidase subunit I (COI), and the sequence of the COI gene could be determined directly. Sequences obtained from the exuvial samples were used to further find similarities within DDBJ/EMBL/GenBank. Genomic DNA from the samples was successfully isolated, and we identified the species. This process suggests that exuvia provides a good sample for extracting DNA from endangered diving beetle larvae without killing them.     

Protective effects of bacteriophage on experimental Lactococcus garvieae infection in yellowtail.

The present study describes the in vitro and in vivo survival of Lactococcus garvieae bacteriophages and the potential of the phage for controlling experimental L. garvieae infection in yellowtail. Anti-L. garvieae phages persisted well in various physicochemical (water temperature, salinity, pH) and biological (feed, serum and alimentary tract extracts of yellowtail) conditions, except for low acidity. In the in vivo, the phage PLgY-16 was detected in the spleens of yellowtail until 24 h after intraperitoneal (i.p.) injection, or the phage was recovered from the intestine of yellowtail 3 h after the oral administration of phage-impregnated feed but undetectable 10 h later. Simultaneous administration of live L. garvieae and phage enhanced recovery of the phage from the spleen or intestine. The survival rate was much higher in yellowtail that received i.p. injection of the phage after i.p. challenge with L. garvieae, compared with that of control fish without phage injection. When fish were i.p. injected with phage at different hours after L. garvieae challenge, higher protective effects were demonstrated in fish that received phage treatment at the earlier time. Protection was also obtained in yellowtail receiving phage-impregnated feed, in which fish were challenged by an anal intubation with L. garvieae. Anal-intubated L. garvieae were detected constantly in the spleens of the control fish, while they were detected sporadically and disappeared from the phage-treated fish 48 h later. On the other hand, orally administered phage was detected at high plaque-forming units from the intestines and spleens of the phage-treated fish until 48 h later. These results indicate that intraperitoneally or orally administered anti-L. garvieae phage prevented fish from experimental L. garvieae infection, suggesting potential use of the phage for controlling the disease.     

Effective DNA extraction method for fragment analysis using capillary sequencer of the kelp, Saccharina

Deoxyribonucleic acid (DNA) fragment analysis can become an effective tool to study genetic differences between species and individuals on saccharinan kelp from which the little genetic diversity has been reported. Here, extraction methods of DNA suitable for use in analysis with a capillary sequencer is examined on Saccharina japonica var. diabolica which contains abundant polysaccharide. When amplified fragment length polymorphism was performed using genomic DNA extracted by seven different methods: (1) commercial kit, (2) original cetyl trimethylammonium bromide (CTAB) method, (3)–(5) three types of modified CTAB method, (6) modified sodium dodecyl sulfate (SDS) method, (7) combination of CTAB method and SDS method, a high reproducible peak that was suitable for analysis was noticeable in the electropherogram in the experiment with the last combination method (7). It is considered that the pretreatment washing of polysaccharide and the subsequent purification for protein and ribonucleic acid in SDS method and for polysaccharide in CTAB method are effective to obtain the high-purity DNA.     

The transformation-sensitive heat shock protein (hsp47) binds specifically to Fetuin

The transformation-sensitive heat shock protein of Mr = 47,000 (hsp47) has been shown to bind to collagen and gelatin. We examined the binding specificity of hsp47. The binding of hsp47 to gelatin-Sepharose 4B was competitively inhibited by fetuin as effectively as by gelatin or collagen, whereas a variety of other proteins tested had no effect. Fetuin-coupled Sepharose 4B was found to bind hsp47 even at high ionic strength, but the complex was dissociated at pH less than or equal to 5.5.)     

Amino Acid Requirements of Two Hyperthermophilic Archaeal Isolates from Deep-Sea Vents, Desulfurococcus Strain SY and Pyrococcus Strain GB-D

Two sulfur-dependent hyperthermophilic archaea, Desulfurococcus strain SY and Pyrococcus strain GB-D, which were isolated from deep-sea hydrothermal vents, utilized free amino acids and peptides obtained from various molecular size fractions of yeast extract. It was found that 11 amino acids were essential for growth. The metabolic products were acetate, i-butyrate, and i-valerate.