Transepithelial transport of chlorogenic acid, caffeic acid, and their colonic metabolites in intestinal caco-2 cell monolayers

Both chlorogenic and caffeic acids exhibited nonsaturable transport in Caco-2 cells, whereas caffeic acid also showed proton-coupled polarized absorption. Thus, the absorption efficiency of caffeic acid was greater than that of chlorogenic acid. Polarized transport of caffeic acid was inhibited by substrates of MCT such as benzoic and acetic acids. Almost all of the apically loaded chlorogenic and caffeic acid was retained on the apical side, and the transepithelial flux was inversely correlated with the paracellular permeability of Caco-2 cells. These results indicate that transport was mainly via paracellular diffusion, although caffeic acid was absorbed to a lesser extent by the monocarboxylic acid transporter (MCT). Furthermore, m-coumaric acid and 3-(m-hydroxyphenyl)propionic acid, the main metabolites of chlorogenic and caffeic acid by colonic microflora, competitively inhibited the transport of fluorescein, a known substrate of MCT. This suggests that their absorption could also be mediated by MCT. These findings have exemplified the physiological importance of MCT-mediated absorption in both phenolic acids per se and their colonic metabolites.     

Changes in the bovine whey proteome during the early lactation period

To investigate time‐dependent change in the bovine whey proteome during the early lactation period, a two‐dimensional gel‐based approach was used in this study. Milk samples were collected from five healthy Friesian‐Holstein dairy cows up to 10 days postpartum. Spot patterns of whey proteins varied drastically from immediately after parturition to 48 h, but no significant changes occurred thereafter. Protein identification by mass spectrometry revealed that the ratios of caseins and immunoglobulins drastically decreased during 48 h postpartum, while those of lower molecular mass proteins such as α‐lactalbumin and β‐lactoglobulin increased. More than 100 spots were detected, being much more abundant in colostral whey than in mature milk whey. Of a total of 25 proteins identified, four, viz. zinc‐α‐2‐glycoprotein, vitamin D‐binding protein, immunoglobulin G2 chain C and β2‐microglobulin, were detectable only in colostrum. Our results indicate that most of the minor whey proteins in colostrum relate to the passive immunity of newborn calves, but some of them play significant roles in nutritional supplementation of the neonate. The characteristics of whey proteins in transition imply that enhancement of innate immunity becomes more important than protection of the neonate against pathogens via passive immunity after 48 h postpartum.     

Structural determination of the oligosaccharides in the milk of a giant anteater (Myrmecophaga tridatyla)

There appears to be strong evidence supporting four major clades of placental mammals: (i) Afrotheria; (ii) Xenarthea; (iii) Euarchontoglires; and (iv) Laurasiathera. This is the first study in which the carbohydrates have been analyzed in the milks of Xenarthea. Our aim was to clarify whether the saccharides of giant anteater milk resemble those of other eutherian species, especially of the Asian elephant, which is one of the Afrotherea. We found that lactose is a dominant saccharide in this milk, as it is in that of most eutherians, and that the milk contains smaller amounts of oligosaccharides, whose structures were determined as Gal(α1‐3)Gal(β1‐4)Glc (isoglobotriose), Neu5Ac(α2‐6)Gal(β1‐4)Glc (6'‐N‐acetylneuraminyllactose), Neu5Gc(α2‐6)Gal(β1‐4)Glc (6'‐N‐glycolylneuraminyllactose), Neu5Ac(α2‐6)Gal(β1‐4)GlcNAc(β1‐3)Gal(β1‐4)Glc (LST c) and Neu5Gc(α2‐6)Gal(β1‐4)GlcNAc(β1‐3)Gal(β1‐4)Glc. This pattern of milk carbohydrates is rather close to that of the milks of other eutherian species. It is notable that the milk contained oligosaccharides with Neu5Gc residues but no oligosaccharides containing α(2‐3) linked Neu5Ac residues such as Neu5Ac(α2‐3)Gal(β1‐4)Glc.     

Knockout mouse production assisted by Blm knockdown

Production of knockout mice using targeted embryonic stem cells (ESCs) is a powerful approach for investigating the function of specific genes in vivo. Although the protocol for gene targeting via homologous recombination (HR) in ESCs is already well established, the targeting efficiency varies at different target loci and is sometimes too low. It is known that knockdown of the Bloom syndrome gene, BLM, enhances HR-mediated gene targeting efficiencies in various cell lines. However, it has not yet been investigated whether this approach in ESCs is applicable for successful knockout mouse production. Therefore, we attempted to answer this question. Consistent with previous reports, Blm knockdown enhanced gene targeting efficiencies for three gene loci that we examined by 2.3–4.1-fold. Furthermore, the targeted ESC clones generated good chimeras and were successful in germline transmission. These data suggest that Blm knockdown provides a general benefit for efficient ESC-based and HR-mediated knockout mouse production.     

Serum iron concentration is candidate inflammatory marker for respiratory diseases in beef cows

We hypothesized that the serum iron (Fe) concentration in cows with respiratory diseases is a satisfactory substitute for major inflammatory markers such as haptoglobin (HPT) and serum amyloid A (SAA). Twenty Japanese Black cows aged 279.6 ± 120.0 days were enrolled, and divided into respiratory diseases and control groups based on the presence of clinical findings of respiratory diseases. As a result, area under the receiver operating characteristic curves for plasma HPT, SAA and serum Fe concentrations for respiratory disease-associated systemic inflammation were excellent, at 1.00, 0.96 and 0.97, respectively. Therefore we confirmed that the serum Fe concentration is a satisfactory substitute for HPT and SAA in beef cows with respiratory diseases.     

Transgene manipulation in rainbow trout using Cre recombinase

Cre/loxP-mediated cell targeting is considered to be a powerful tool for biotechnology in farmed fish. As a first step toward establishing cell targeting in salmonids, we analyzed the functionality of the Cre/loxP system in rainbow trout. We first established stable transgenic strains carrying the DsRed gene, which was flanked by loxP sites and further spliced with the EGFP gene. By microinjecting Cre complementary RNA (cRNA) into fertilized eggs of the transgenic trout, the functionality of the Cre/loxP system was evaluated. The results showed that all of the embryos exhibited green fluorescence in at least some of their cells. While 19 out of 20 embryos comprised cells showing both green and red fluorescence, the remaining embryo showed only green fluorescence. Polymerase chain reaction (PCR) using primers designed to recognize sequences outside of the two loxP sites revealed that, in addition to long intact fragments, the 19 individuals carried short fragments that were equivalent in length to the loxP-excised fragments. The remaining green embryo carried only this short fragment. DNA sequencing of the short fragment revealed that it lacked the DNA fragments flanking the loxP sites and the spliced fragments did not contain any sequence rearrangements. These results suggest that the Cre/loxP system is functional in rainbow trout.     

Virulence of infectious pancreatic necrosis virus is associated with the larger RNA segment (RNA segment A)

Abstract. Infectious pancreatic necrosis virus (IPNV) contains two segments of double- stranded RNA (the larger RNA segment, A; and a smaller RNA segment, B). The IPNV- Buhl isolate belongs to the VR299 serotype, and eel virus European (EVE) is serologically the same as the Ab serotype. IPNV-Buhl is virulent to rainbow trout fry, Oncorhynchus mykiss (Walbaum), whereas EVE is avirulent. To determine the genetic basis for the difference of the virulence, intertypic reassortants were made between IPNV-Buhl and EVE. The reassortants with the genotypes of P/E (segment A of IPNV-Buhl and segment B of EVE) and E/P (segment A of EVE and segment B of IPNV-Buhl) were studied for their pathogenicity to rainbow trout fry. The P/E reassortant was highly virulent, similar to the IPNV-Buhl parent (P/P), while the E/P reassortant was avirulent, as was the EVE parent (E/E). A high level of viral replication was observed in almost all organs of the fry infected with the P/E reassortant and the IPNV-Buhl parent. In contrast, viral replication was restricted to the epidermal cells of the fry infected with the E/P reassortant and the EVE parent. The results demonstrate that the larger RNA segment (RNA segment A) of IPNV is related to cell tropism in vivo and is responsible for virulence in rainbow trout fry.     

Microbial biomass and nitrogen transformations in surface soils strongly acidified by volcanic hydrogen sulfide deposition in Osorezan, Japan

Volcanic acidification has created unique ecosystems that have had to adapt to the acidic environments in volcanic regions. To characterize the primary microbial properties of strongly acidified soils in such environments, we investigated microbial biomass, nitrogen transformations and other relevant chemical properties in the surface soils of solfatara and forests from Osorezan, a typical volcanic region in Japan, and compared the results to common Japanese forest soils. Soil microbial biomass C (MBC) and N (MBN) were determined using the chloroform fumigation–extraction method. Potential net N mineralization and net nitrification were measured in aerobic laboratory incubations. Long-term acidification in the Osorezan soils by volcanic hydrogen sulfide deposition caused low soil pH (3.0–3.8), base cation deficiency and increased concentrations of toxic ions such as Al³⁺. The proportions of MBC to total carbon (MBC/TC ratio) and MBN to total nitrogen (MBN/TN ratio) were lower than those in common Japanese forest soils. The extreme acidic conditions may have inhibited microbial survival in the Osorezan acid soils. Net N mineralization occurred at rates comparable to those in common Cryptomeria japonica forest soils, probably because of the presence of acid-tolerant soil microorganisms. Net nitrification was completely inhibited and autotrophic ammonia oxidizers were not detected by the MPN method. The inhibition of nitrification prevents nitrogen leaching from the soils, thus maintaining a nitrogen cycle in the volcanic acid region in which     (and NH₃) is recycled among microorganisms and plants.