Succinate accumulation in pig large intestine during antibiotic-associated diarrhea and the constitution of succinate-producing flora

Succinate was the major organic acid detected in the hindgut content of pigs suffering from antibiotic-associated diarrhea. Antibiotic-associated diarrhea was induced by an oral dose of polymyxin B sulfate (3,000,000 units/day) or an intramuscular injection of enrofloxacin (0.6 g enrofloxacin/day). In the large intestine of enrofloxacin-treated pigs, Gram-negative facultative anaerobic rods phylogenetically related to Escherichia coli and Gram-positive facultative anaerobic non-spore-forming rods phylogenetically related to Lactobacilli were isolated as succinate producers. Succinate-producing Lactobacilli were only isolated as the succinate producer in polymyxin B sulfate-treated pigs. In contrast to antibiotic-associated diarrhea pigs, bacteria belonging to Bacteroidaceae, Fusobacteria, and Enterobacteriaceae were detected as succinate producers in a non-treated pig. In antibiotic-associated diarrhea conditions, antibiotic-resistant Enterobacteria, E. coli in particular, and Lactobacilli may contribute to an abnormal succinate accumulation and may affect water absorption in the hindgut that relates to an expression of antibiotic-associated diarrhea.     

Sorting specificity of spermatogenic cell specific region of mouse hexokinase-s (mHk1-s)

Hexokinase is the first enzyme involved in the glycolysis process that produces glucose phosphorylate. Our previous study reported on our cloning of mouse Hk1-s (mHk1-s) cDNA, which were expressed only in testis cells, and noted that this cDNA has a spermatogenic cell-specific region (SSR) that replaces the porin binding domain (PBD) in the Hk1of somatic cells. Although we know that PBD binds to the outer membrane of a mitochondrion, the role of the SSR is not yet understood. To investigate the intracellular localization of SSR, we constructed expression vectors with the epitope tag (GFP-, HA-), subcloned SSR, or PBD cDNA. We transfected these vectors in mouse fibroblast, NIH3T3 cells, after which we observed the localization of the SSR and PBD in the NIH3T3 cells. Our current study using the immunocytochemical method revealed that PBD is concentrated around the mitochondrion. However, the SSR could not be ascribed to the mitochondrion, ER, or nuclear colocalization. Moreover, subcellular fractionation analysis showed that PBD was detected in the mitochondrial fraction, and that SSR was detected in the cytosolic fraction. Our findings suggest that PBD of Hk1 targets mitochondrion, but the SSR of mHk1-s targets some specific organellae.     

Cytotoxic screening of medicinal and edible plants in Okinawa,Japan, and identification of the main toxic constituent of Rhodea japonica (Omoto)

The cytotoxic activity of ethanol extracts from 53 parts of 36 species of medicinal and edible plants cultivated in Okinawa was measured by using K562 human leukemia cells by a flow cytometric method. Two extracts from Rhodea japonica and Hypericum chinense were cytotoxic at a concentration of 10 microg/ml. The main cytotoxic constituent of Rhodea japonica was isolated and identified to be rhodexin A, which has been isolated as a cardetonic agent of the plant. The IC(50) value for rhodexin A against the growth of K562 cells was 19 nM, this activity being much stronger than that of ouabain (IC(50), 60 nM).     

Effects of animal or plant protein diets on cecal fermentation in guinea pigs (Cavia porcellus), rats (Rattus norvegicus) and chicks (Gallus gallus domesticus)

Monogastric herbivores such as the guinea pig depend on energy supply from enteric fermentation as short-chain fatty acids (SCFA) corresponding to 30-40% of their maintenance energy requirements. They evolved specific digestive system to adapt their indigenous microflora to plant polysaccharides fermentation. No information has been available about the adaptability of microbial fermentation in hindgut of the monogastric herbivorous to an animal protein diet. We investigated if the guinea pig can fully retrieve energy of an animal protein diet by hindgut fermentation compared with a plant protein diet. For comparison, we also studied two omnivores. End products of in vitro cecal fermentation (SCFA, ammonia and gases) were measured to judge how well an animal protein diet could be fermented. The animal protein diet resulted in the less intensive fermentation with increased feed intake and volume of cecal contents than the plant protein diet only in guinea pigs. This may be due to a limited capacity of the hindgut microflora to adapt to the substrate rich in animal protein. We also found that chick cecal contents produced methane at higher emission rate than ruminants.     

Milk and dairy products in cancer prevention: focus on bovine lactoferrin

Milk and dairy products constitute an important part of the western style diet. A large number of epidemiological studies have been conducted to determine effects of consumption on cancer development but the data are largely equivocal, presumably reflecting the different included components. It has been proposed that whereas fats in general could promote tumor development, individual milk fats like conjugated linoleic acid could exert inhibitory effects. There is also considerable evidence that calcium in milk products protects against colon cancer, while promoting in the prostate through suppression of circulating levels of 1,25-dihydroxyvitamin D3. Whey protein may also be beneficial, as shown by both animal and human studies, and experimental data have demonstrated that the major component bovine lactoferrin (bLF), inhibits colon carcinogenesis in the post-initiation stage in male F344 rats treated with azoxymethane (AOM) without any overt toxicity. The incidence of adenocarcinomas in the groups receiving 2% and 0.2% bLF were thus 15% and 25%, respectively, in contrast to the 57.5% control value (P<0.01 and P<0.05, respectively). Results in other animal models have provided further indications that bLF might find application as a natural ingredient of milk with potential for chemoprevention of colon and other cancers.     

Simultaneous Detection of Both Single Nucleotide Variations and Copy Number Alterations by Next-Generation Sequencing in Gorlin Syndrome

Gorlin syndrome (GS) is an autosomal dominant disorder that predisposes affected individuals to developmental defects and tumorigenesis, and caused mainly by heterozygous germline PTCH1 mutations. Despite exhaustive analysis, PTCH1 mutations are often unidentifiable in some patients; the failure to detect mutations is presumably because of mutations occurred in other causative genes or outside of analyzed regions of PTCH1, or copy number alterations (CNAs). In this study, we subjected a cohort of GS-affected individuals from six unrelated families to next-generation sequencing (NGS) analysis for the combined screening of causative alterations in Hedgehog signaling pathway-related genes. Specific single nucleotide variations (SNVs) of PTCH1 causing inferred amino acid changes were identified in four families (seven affected individuals), whereas CNAs within or around PTCH1 were found in two families in whom possible causative SNVs were not detected. Through a targeted resequencing of all coding exons, as well as simultaneous evaluation of copy number status using the alignment map files obtained via NGS, we found that GS phenotypes could be explained by PTCH1 mutations or deletions in all affected patients. Because it is advisable to evaluate CNAs of candidate causative genes in point mutation-negative cases, NGS methodology appears to be useful for improving molecular diagnosis through the simultaneous detection of both SNVs and CNAs in the targeted genes/regions.     

Macrophage inflammatory protein-1α (MIP-1α) enhances a receptor activator of nuclear factor κB ligand (RANKL) expression in mouse bone marrow stromal cells and osteoblasts through MAPK and PI3K/Akt pathways

Osteolytic lesions are rapidly progressive during the terminal stages of myeloma, and the bone pain or bone fracture that occurs at these lesions decreases the patients’ quality of life to a notable degree. In relation to the etiology of this bone destruction, it has been reported recently that MIP-1α, produced in large amounts in myeloma patients, acts indirectly on osteoclastic precursor cells, and activates osteoclasts by way of bone-marrow stromal cells or osteoblasts, although the details of this process remain obscure. In the present study, our group investigated the mechanism by which RANKL expression is induced by MIP-1α and the effects of MIP-1α on the activation of osteoclasts. RANKL mRNA and RANKL protein expressions increased in both ST2 cells and MC3T3–E1 cells in a MIP-1α concentration-dependent manner. RANKL mRNA expression began to increase at 1 h after the addition of MIP-1α; the increase became remarkable at 2 h, and continuous expression was observed subsequently. Both ST2 and MC3T3-E1 cells showed similar levels of increased RANKL protein expression at 1, 2, and 3 days after the addition of MIP-1α. After the addition of MIP-1α, the amount of phosphorylated ERK1/2 and Akt protein expressions showed an increase, as compared to the corresponding amount in the control group. On the other hand, the amount of phosphorylated p38MAPK protein expression showed a decrease from the amount in the control group after the addition of MIP-1α. U0126 (a MEK1/2 inhibitor) or LY294002 (a PI3K inhibitor) was added to ST2 and MC3T3-E1 cells, and was found to inhibit RANKL mRNA and RANKL protein expression in these cells. When SB203580, a p38MAPK inhibitor, was added, RANKL mRNA and RANKL protein expression were increased in these cells. MIP-1α was found to promote osteoclastic differentiation of C7 cells, an osteoclastic precursor cell line, in a MIP-1α concentration-dependent manner. MIP-1α promoted differentiation into osteoclasts more extensively in C7 cells incubated together with ST2 and MC3T3-E1 cells than in C7 cells incubated alone. These results suggested that MIP-1α directly acts on the osteoclastic precursor cells and induces osteoclastic differentiation. This substance also indirectly induces osteoclastic differentiation through the promotion of RANKL expression in bone-marrow stromal cells and osteoblasts. The findings of this investigation suggested that activation of the MEK/ERK and the PI3K/Akt pathways and inhibition of p38MAPK pathway were involved in RANKL expression induced by MIP-1α in bone-marrow stromal cells and osteoblasts. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.     

Effects of leaf quality and microhabitat on the survival of a leaf-rolling weevil (Attelabidae)

Female Cycnotrachelus roelofsi (Coleoptera: Attelabidae) construct two types of leaf roll, i.e., cut-off cradles (CCs) and suspended cradles (SCs), in which to lay eggs; these cradles are generally constructed using older and younger leaves, respectively. We conducted two experiments to determine whether the quality of cradle leaves affects egg and larval survival. In the first experiment, we severed all SCs from a tree and placed them on the ground with unmanipulated CCs in the early breeding season. In the second experiment, we resuspended all CCs in a tree with unmanipulated SCs in the late breeding season. We also compared leaf mass per area (LMA), polyphenol content, and nitrogen content between the two cradle types to determine whether there were any differences in leaf quality. Larval mortality, probably caused by cradle herbivory, was significantly greater in severed SCs than in intact CCs in the early season, suggesting that leaf quality had a profound effect on larval mortality in the terrestrial microhabitat. In contrast, larval mortality did not differ between resuspended CCs and intact SCs in the late season, suggesting that leaf quality had little effect on larval mortality in the arboreal microhabitat. LMA was higher in CCs than in SCs, but there were no differences in the nitrogen and polyphenol contents. These results suggest that cradles constructed using mature, tough leaves were more effective against terrestrial cradle herbivores than those constructed using new, soft leaves.     

Roles of the three L‐domains in β‐retrovirus budding

Retroviral Gag protein plays a critical role during the late stage of virus budding and possesses a so‐called L‐domain containing PT/SAP, PPxY, YxxL or FPIV motifs that are critical for efficient budding. Mason–Pfizer monkey virus (M‐PMV) contains PSAP, PPPY, and YADL sequences in Gag. This study was performed to investigate the roles of these three L‐domain‐like sequences in virus replication in three different cell lines, 293T, COS‐7 and HeLa cells. It was found that the PPxY motif plays an essential role in progeny virus production as a major L‐domain in all three cell lines. The PSAP sequence was shown to function as an additional L‐domain in HeLa cells and to promote efficient release of M‐PMV; however, this sequence was dispensable for M‐PMV production in 293T and COS‐7 cells, suggesting that the role of the PSAP motif as an L‐domain in M‐PMV budding is cell type‐dependent. Viruses possessing multiple L‐domains appear to change the L‐domain usage to replicate in various cells. On the other hand, the YADL motif was required for M‐PMV production as a transport signal of Gag to the plasma membrane, but not as an L‐domain.