Identification of genes required for the fitness of Rhodococcus equi during the infection of mice via signature-tagged transposon mutagenesis

Rhodococcus equi is a Gram-positive facultative intracellular bacterium that causes pyogranulomatous pneumonia in foals and immunocompromised people. In the present study, signature-tagged transposon mutagenesis was applied for the negative selection of R. equi mutants that cannot survive in vivo. Twenty-five distinguishable plasmid-transposon (plasposon) vectors by polymerase chain reaction (PCR), each containing a unique oligonucleotide tag, were constructed and used to select the transposon mutants that have in vivo fitness defects using a mouse systemic infection model. Of the 4,560 transposon mutants, 102 mutants were isolated via a real-time PCR-based screening as the mutants were unable to survive in the mouse model. Finally, 50 single transposon insertion sites were determined via the self-cloning strategy. The insertion of the transposon was seen on the virulence plasmid in 15 of the 50 mutants, whereas the remaining 35 mutants had the insertion of transposon on the chromosome. The chromosomal mutants contained transposon insertions in genes involved in cellular metabolism, DNA repair and recombination, gene regulation, non-ribosomal peptide synthesis, and unknown functions. Additionally, seven of the chromosomal mutants showed a reduced ability to multiply in the macrophages in vitro. In this study, we have identified several biosynthetic pathways as fitness factors associated with the growth within macrophages and survival in mice.     

Effects of anthocyanins on psychological stress-induced oxidative stress and neurotransmitter status

There is strong evidence that oxidative stress participates in the etiology of neurodegenerative diseases such as Parkinson's, and Alzheimer's diseases. Moreover, emotional stress effects in the central nervous system play a vital role in homeostasis. The protective effect of anthocyanins on the cerebral oxidative stress was studied using the whiskers cut model. In mice, such treatment causes psychological or emotional distress leading to oxidative stress in tissues. To investigate the in vivo antioxidant activity of anthocyanins, an extract of Vaccinium myrtillis L., an anthocyanin mixture, was orally administered (100 mg/kg of body weight.) to mice for 7 days, and then psychological stress was assessed by cutting off their whiskers. Whisker removal increased both protein carbonyl formation and lipid peroxidation in the brain, heart, kidney, and liver. Further, the levels of oxidative markers showed regional differences in the brain. Concomitantly, dopamine neurotransmitter levels were altered in both the midbrain and the brain cortex. Orally administered anthocyanins were also active in the brain, suppressing stress-induced cerebral oxidative stress and dopamine abnormalities in distressed mice. These effects of anthocyanin treatment suggest their possible usefulness for the treatment of cerebral disorders related to oxidative stress.     

Dietary phosphatidylinositol prevents the development of nonalcoholic fatty liver disease in Zucker (fa/fa) rats

Recent studies have shown that dietary phospholipids, especially phosphatidylcholine and phosphatidylserine, have various beneficial biological effects. However, there are not enough data concerning the physiological function of dietary phosphatidylinositol (PI). The metabolic syndrome, a cluster of metabolic abnormalities such as dyslipidemia, diabetes mellitus, and hypertension, is a widespread and increasingly prevalent disease in industrialized countries. Nonalcoholic fatty liver disease (NAFLD) is often associated with features of the metabolic syndrome. NAFLD describes the spectrum of liver damage ranging from hepatic steatosis to steatohepatitis, liver fibrosis, and cirrhosis, and it is emerging as the most common liver disease worldwide. The present study examined whether dietary PI protects Zucker ( fa/ fa) rats from the metabolic syndrome. For 4 weeks, rats were fed semisynthetic diets containing either 7% soybean oil or 5% soybean oil plus 2% PI. Dietary PI markedly prevented the development of hepatomegaly and hepatic steatosis and lowered hepatic injury markers in serum. Additionally, hyperinsulinemia was relieved by the feeding of dietary PI in Zucker rats. These effects were attributable to an increase in serum adiponectin, enhancement of fatty acid beta-oxidation, and suppression of mRNA expression of inflammatory genes in the liver. This is the first report that dietary PI increases serum adiponectin level and prevents the development of NAFLD in a rat model of the metabolic syndrome.     

Effect of temperature on the development of eggs and the daily pattern of spawning of round herring <Emphasis Type="Italic">Etrumeus teres</Emphasis>

Effect of temperature on the development of eggs of round herring Etrumeus teres was experimentally examined to construct a temperature-dependent egg development model. Mature fish were collected in the field and their eggs were artificially fertilized onboard. The eggs were incubated at nine temperatures set between 14.0 and 25.0°C. All eggs at the lowest three temperatures, 14.0°C, 15.0°C, and 16.0°C, ceased development and died at various stages before hatching. Durations required to hatching after fertilization ranged from 38.0 h at 25.0°C to 90.0 h at 17.5°C. The temperature-dependent egg development model, i.e., egg age in hours (y _i,t ) at the ith stage and temperature t (°C), was expressed as: y _i,t  = 4.604 × exp(−0.100 × t −0.129 × i) × i ^2.593. From the application of the model to early-stage eggs collected in the field, it is concluded that round herring starts spawning immediately after sunset and almost completes spawning by midnight. The temperature-dependent egg development model and the daily pattern of spawning presented in this study are essential tools for developing the daily egg production method to estimate the spawning stock biomass.     

Effect of temperature decrease on oocyte development,sex steroids,and gonadotropin β-subunit mRNA expression levels in female Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

To improve understanding of the mechanism of early ovarian development in eels, the effects of water temperature decrease on oocyte development, plasma levels of sex steroids [estradiol 17β (E2), testosterone (T), 11-ketotestosterone (11-KT)], and gonadotropin β-subunit [follicle-stimulating hormone (FSHβ), luteinizing hormone (LHβ)] messenger RNA (mRNA) expression levels were investigated. A total of 27 female Japanese eels Anguilla japonica were divided into initial, control, and test (water temperature decrease) groups. Starting on 22 September 2009, eels in the test group were reared in a tank with gradual temperature decrease from 25°C to 15°C over 39 days, while the control group was maintained at 25°C. The test group accumulated more oil droplets in their oocytes than did the other groups. Levels of sex steroids, especially 11-KT, were higher in the test group. In contrast, FSHβ and LHβ mRNA expression levels were lower in the test group. These results suggest that water temperature decrease only induced an early stage of ovarian development that was partly affected by an 11-KT increase. For further maturation, other environmental factors related to induction of gonadotropin increase appear to be needed.     

Pythium rot of chingensai ( Brassica campestris L. chinensis group) caused by Pythium ultimum var. ultimum and Pythium aphanidermatum

Severe rot was found at the base of leaves and stems of chingensai (Brassica campestris L. chinensis group) in Okayama Prefecture in 2000. The causal fungi were morphologically identified as Pythium ultimum Trow var. ultimum and P. aphanidermatum (Edson) Fitzpatrick. This is the first report of rot caused by Pythium species on chingensai. We named this disease Pythium rot of chingensai.     

Occurrence of blood disease of banana in Sumatra,Indonesia

Since 1991, the sudden death of cultivated banana plants has been widely observed in the southern region of Sumatra Island, Indonesia. Wilting from loss of petiole and midrib turgidity, yellowing, and necrosis of leaves was followed by death of the whole plant. Reddish brown bacterial ooze exuded from the cut surface of infected pseudostems and fruits. The colony appearance of the isolated bacterium was similar to that of Ralstonia solanacearum. The bacterium was pathogenic to banana plants but not to tomato. Its bacteriological properties agreed with those of blood disease bacterium (BDB) of banana described previously. The 16S rDNA sequence of strain Banana E had conserved bases characteristic of BDB. Based on these results, the causal agent was identified as BDB, which is a close relative of Ralstonia. The isolates have resistance against antibiotics, such as chloramphenicol and tetracycline.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB095535     

Loss of milk yield due to Akabane disease in dairy cows

Akabane disease is an infection with clinical signs of congenital malformation and abortion in ruminants. Abnormal parturitions caused by Akabane disease result in great economic loss. The purpose of this study is to estimate the reduction in the milk yield from abnormal parturition due to Akabane disease. The data were collected from 33 Holstein cows on 11 farms. The animals had abnormal parturitions during the period from September 1998 to March 1999, and were diagnosed as having Akabane disease. The mean and standard deviation of the rate of reduction in the milk yield of 33 cows after abnormal parturition caused by Akabane disease was -11.4 +/- 14.9%. The means and standard deviations of the rate of reduction of four cows calving abnormally at 220-239 days of gestation, nine cows calving abnormally at 240-270 days of gestation, and 20 cows calving abnormally at 271-300 days of gestation were -26.6 +/- 24.7%, -14.7 +/- 11.0%, and -6.9 +/- 12.3%, respectively. In this study, we demonstrated that the rate of reduction in the milk yield in cows affected by Akabane disease was -11.4 +/- 14.9%, but values as high as -26.6 +/- 24.7% were reached in the comparison with the milk yield obtained after normal parturition.