Identification of marker genes for intestinal immunomodulating effect of a fructooligosaccharide by DNA microarray analysis

Prebiotic fructooligosaccharides are noted for their intestinal immunodulating effects, and the identification of markers for the effects is a matter of great concern. This study aimed to identify marker genes for physiological effects of a particular fructooligosaccharide (FOS) on a host animal and also to define the target of its function in the small intestine. DNA microarray technology was used to screen candidate marker genes, and comprehensive changes in gene expressions in the ileum of mice fed with FOS were investigated. One of the major physiological effects of FOS was intestinal immunomodulation. Marker genes were then identified for major histocompatibility complex classes I and II, interferon, and phosphatidylinositol metabolites. Also, the ileum was segmented into Peyer's patch (PP) and the other ileal organ (DeltaPP), and these were analyzed by quantitative RT-PCR method, with the result that the site for recognizing the FOS function was the DeltaPP rather than the PP. This is the first paper showing the markers for the physiological effects of FOS in the small intestine at gene expression level. Applying these marker genes would make it possible to clarify the mechanisms of how the administration of dietary FOS and associated changes in the intestinal environment are recognized by host organisms as well as how its immunomodulating effects are expressed in the body.     

Two Resistance Modes to Clover yellow vein virus in Pea Characterized by a Green Fluorescent Protein-Tagged Virus

ABSTRACT This study characterized resistance in pea lines PI 347295 and PI 378159 to Clover yellow vein virus (ClYVV). Genetic cross experiments showed that a single recessive gene controls resistance in both lines. Conventional mechanical inoculation did not result in infection; however, particle bombardment with infectious plasmid or mechanical inoculation with concentrated viral inocula did cause infection. When ClYVV No. 30 isolate was tagged with a green fluorescent protein (GFP) and used to monitor infection, viral cell-to-cell movement differed in the two pea lines. In PI 347595, ClYVV replicated at a single-cell level, but did not move to neighboring cells, indicating that resistance operated at a cell-to-cell step. In PI 378159, the virus moved to cells around the infection site and reached the leaf veins, but viral movement was slower than that in the susceptible line. The viruses observed around the infection sites and in the veins were then recovered and inoculated again by a conventional mechanical inoculation method onto PI 378159 demonstrating that ClYVV probably had mutated and newly emerged mutant viruses can move to neighboring cells and systemically infect the plants. Tagging the virus with GFP was an efficient tool for characterizing resistance modes. Implications of the two resistance modes are discussed.     

Temporal and topographic profiles of tissue hypoxia following transient focal cerebral ischemia in rats

Intravascular accumulation of blood cells after brain ischemia-reperfusion can cause obstruction of cerebral blood flow and tissue hypoxia/ischemia as a consequence. In the present study, we examined temporal and topographic changes of tissue hypoxia/ischemia after occlusion of the middle cerebral artery (MCA) for 60 min in rats with immunohistochemical staining for hypoxia (2-nitroimidazole hypoxia marker: hypoxyprobe-1 adducts). Our results showed that tissue hypoxia expressed as positive staining for hypoxyprobe-1 adducts preceded neuronal degeneration. Platelets and granulocytes were detected close to the hypoxyprobe-1 adducts positive area. These results suggested that the hypoxic environment could persist even after reperfusion of MCA, because of vascular obstruction with accumulation of platelets and granulocytes.     

Feeding Aspergillus awamori reduces skeletal muscle protein breakdown and stimulates growth in broilers

This study was conducted to show that dietary supplementation of a fungus, Aspergillus awamori called Koji in Japan, reduces skeletal muscle protein breakdown and stimulates growth in broiler chickens. A total of 30 chicks at 15 days of age was divided into control and two treatment groups (10 birds per treatment). Control group was fed basal diet and treatment groups were fed the basal diets supplemented with A. awamori at levels of 0.05% and 0.2%. The birds were raised for 12 days from 15 to 27 days of age and then the effect on growth, organ weights and plasma 3‐methylhistidine concentration and digestibilities of protein and energy was evaluated. The messenger RNAs (mRNAs) of atrogin‐1, ubiquitin, proteasome, m‐calpain, µ‐calpain, β‐actin, myosin and pax‐7 in the breast muscle were also measured. Body weight gain and breast muscle weight were increased, although feed intake was decreased by the fungus and thus feed efficiency was increased. Protein and energy digestibilities were increased. Furthermore, plasma 3‐methylhistidine concentration was decreased by the fungus. The mRNAs of atrogin‐1, ubiquitin, proteasome, m‐calpain and µ‐calpain were all decreased. The mRNA of β‐actin but not myosin and pax‐7 was slightly increased by the fungus. In conclusion, feeding A. awamori improves growth performance because skeletal muscle proteolytic activity is reduced and digestibilities of energy and protein are increased.     

<Emphasis Type="Italic">White clover mosaic virus</Emphasis>-induced gene silencing in pea

Double-stranded RNAs formed in secondary structures and replicative intermediates of viral genomes are thought to strongly elicit RNA silencing. This phenomenon is known as virus-induced gene silencing (VIGS). VIGS is a powerful tool for modifying gene expression in host plants. We constructed a virus vector based on White clover mosaic virus (WClMV) and demonstrated VIGS of phytoene desaturase (PDS) in pea. Photobleaching of tissues, caused by VIGS of PDS, was observed in restricted areas of upper leaves and stems. We confirmed that the PDS mRNA and subgenomic RNAs of WClMV were reduced in the photobleached tissues.     

CD38 gene disruption inhibits the contraction induced by alpha-adrenoceptor stimulation in mouse aorta

CD38 is an ectoenzyme with ADP-ribosyl cyclase and hydrolase activities, which synthesizes cyclic ADP-ribose from NAD and hydrolyzes cyclic ADP-ribose to ADP-ribose. It has been shown that cyclic ADP-ribose is a potent Ca(2+) mobilizing messenger in many cells. To know the physiological role of cyclic ADP-ribose in vascular smooth muscle, we examined the effects of various agonists in the aorta isolated from CD38 knockout (CD38(-/-)) mouse. Western blot analysis showed that CD38 protein was detected in the aorta isolated from wild-type (CD38(+/+)) mouse, but not from CD38(-/-) mouse. In the aortae isolated from both CD38(+/+) and CD38(-/-) mice, KCl, phenylephrine and norepinephrine induced concentration-dependent contraction. KCl produced similar concentration-dependent responses in the aortae from both CD38(+/+) and CD38(-/-) mice. Maximum force of contraction induced by KCl (65 mM) was same in the size. Phenylephrine- and norepinephrine-induced contractions were, however, significantly smaller in the aortae from CD38(-/-) mice than in those from CD38(+/+) mice. 5-Hydroxytryptamine, endothelin-1, caffeine and thapsigargin-induced contractions were not significantly different in these two aortae. These results suggest that CD38 gene disruption inhibits alpha-adrenoceptor-induced vascular contractions and cyclic ADP-ribose-mediated signal transduction system is committed in these responses.     

Positional effect of gene insertion on genetic stability of a clover yellow vein virus-based expression vector

The stability of the inserted genes in the viral expression vector varied depending on the sequence introduced and the position of insertion. Infectious cDNA to Clover yellow vein virus (pClYVV) was modified to insert a foreign gene at two independent sites: one, along with a polylinker, between the NIb and CP genes (pClYVV/CP/W) and the other between P1 and HC-Pro (pClYVV-Pst/CP). The green fluorescent protein (GFP) gene was inserted into either pClYVV/CP/W or pClYVV-Pst/CP. GFP gene was stably maintained and expressed in both vectors following serial passages in plants. Progeny viruses from both constructs accumulated in similar amounts and at rates of 70%–80% of that of the wild-type virus. On the other hand, progeny viruses carrying the human interferon- (hIFN) gene cloned in pClYVV-Pst/CP were genetically unstable owing to frequent deletions of the cloned gene during passage through plants. In contrast, the hIFN sequence cloned in pClYVV/CP/W was stably maintained in viruses after several passages in broad bean plants, and the progeny virus accumulated at the rate of about 50%–100% of that of the wild-type virus. The nucleotide sequence analyses indicated that the genetic instability of the inserted sequence results from homologous recombination of viral vector and inserted DNA sequences; it is not due to the inserted sequence alone.     

Increase in apoptotic polymorphonuclear neutrophils in peripheral blood after intramammary infusion of Escherichia coli lipopolysaccharide

A transient increase in apoptotic polymorphonuclear neutrophils (PMNs) as revealed by the terminal deoxynucleotidyl, transferase-mediated dUTP nick end labeling (TUNEL) technique in bovine jugular and milk vein blood was observed 4 h after intramammary infusion of Escherichia coli lipopolysaccharide (LPS) (jugular vein; before infusion 10.1%, 4h 58.3%: milk vein; before infusion 13.2%, 4 h 76.6%) decrease in PMA-induced oxidative bursts of PMNs was also observed during the same period and continued until 8 h after the infusion. TUNEL-positive cells showed an intention of a Comet tail as detected by a single-cell gel electrophoresis assay (Comet assay) and the morphological apoptotic future, though DNA fragmentation was not clearly detected. A definite decrease in peripheral PMNs and a marked increase in PMNs in the LPS-infused teat cistern were observed during the same period. The migration of milk vein blood-derived PMN and the expression of adhesion receptors (L-selectin and CD18) on PMN were suppressed, accompanied by an increase in apoptotic cells. TUNEL-positive PMN observed in normal animals showed a reduced migration capacity. The increase in apoptotic PMNs observed in the LPS-infused cattle was thought to be due to the remaining intravenous spontaneous apoptotic cells existing under the normal condition (the aging cell), and this increase appeared to lower the expression of adhesion receptors and the migration capacity. Decreased PMA-induced oxidative burst activity in PMN was thought to be derived from these aging cells and immature band cells appearing in the circulation as a subsequent event of leukopenia and/or severe stress associated with mastitis. The results from the present study indicate the possibility that the function of PMN in the circulation at early stages of bovine mastitis is regulated by the kinetics of PMN aging.     

Effects of food and water withdrawal and high temperature exposure on diurnal variation in blood viscosity of broiler chickens

1. Three experiments were conducted to investigate the diurnal variation of blood viscosity in broilers. In experiment 1 food and water were supplied freely at 20C (20-FW). In experiment 2 food and water were withdrawn at 20C (20-NFW), while in experiment 3 food and water were withdrawn at 30C (30-NFW). 2. Blood sampling time points were 09.00 h, 15.00 h, 21.00 h, 03.00 h and 09.00 h the next day in each experiment. 3. In all experiments, whole blood viscosity (WBV), red blood cell count (RBC) and haematocrit (HCT) were greater during the dark (21.00 h and 03.00 h) than during the light period. During the dark period, there were no differences in WBV, RBC and HCT between 20-FW and 20-NFW, or between 20-NFW and 30-NFW. At 09.00 h, WBV and HCT were higher in 20-FW than in 20-NFW. At 15.00 h and 09.00 h (day 2), WBV and HCT were greater in 20-NFW than in 30-NFW. 4. There were no light-dark differences in plasma viscosity (PV), plasma protein concentration (PPC) or mean corpuscular volume (MCV) in any experiment. However, 20-NFW birds had a lower PPC and higher MCV compared with 20-FW, and a higher PPC and lower MCV compared with 30-NFW, while no difference was found in PV. 5. WBV increased linearly with RBC and HCT. PV increased with PPC, while MCV decreased. 6. These results indicate that there is diurnal variation in whole blood viscosity, which is greater during the dark than during the light period. During the light period it is strongly influenced by high environmental temperature and food and water withdrawal. C