Detection and differentiation of Fusarium oxysporum f. sp. lycopersici race 1 using loop-mediated isothermal amplification with three primer sets.

UNLABELLED: Fusarium oxysporum f. sp. lycopersici (Fol) causes tomato wilt. Based on the difference in pathogenicity towards tomato cultivars, Fol is classified into three races. In this study, a rapid method is developed for the detection and discrimination of Fol race 1 using a loop-mediated isothermal amplification (LAMP) assay with two primer sets targeting a region of the nucleotide sequence of the SIX4 gene specific for race 1 and a primer set targeting the SIX5 gene, conserved in all known Fol isolates. Upon LAMP reaction, amplification using all three primer sets was observed only when DNA of Fol race 1 was used as a template, and not when DNA of other Fol races or other fungal species was used. This method could detect 300 fg of Fol race 1 DNA, a 100-fold higher sensitivity than that obtained by conventional PCR. The method can also detect DNA extracted from soil artificially infested with Fol race 1. It is now possible to detect Fol race 1 in colonies and infected tomato stems without DNA isolation. This method is a rapid and simple tool for discrimination of Fol race 1. SIGNIFICANCE AND IMPACT OF THE STUDY: This study developed a loop-mediated isothermal amplification (LAMP) assay for detection and differentiation of Fusarium oxysporum f. sp. lycopersici (Fol) race 1 by using three primer sets targeting for the SIX4 and SIX5 genes. These genes are present together only in Fol race 1. This method can detect Fol race 1 in infected tomato stems without DNA extraction, affording an efficient diagnosis of Fusarium wilt on tomatoes in the field.

Leakage of energy to the body surface during defibrillation shock by an implantable cardioverter-defibrillator (ICD) system--experimental evaluation during defibrillation shocks through the right ventricular lead and the subcutaneous active-can in canines.

The leakage of electrical current to the body surface during defibrillation shock delivery by an implantable cardioverter-defibrillator (ICD) device (the Medtronic Jewel Plus PCD system) was evaluated in 5 dogs. The defibrillation shocks were delivered between the active-can implanted in the left subclavicular region and the endocardial lead placed in the right ventricle at the energy levels of 1, 2, 8, 12, 24 and 34 J. During each delivery, the electrical current leakage from the body surface was measured by electrodes connected to a circuit at 4 recording positions: (A) parallel-subcutaneous (the electrodes were fixed in the subcutaneous tissue of the left shoulder and the right lower chest, and the direction of the electrode vector was parallel to the direction of the defibrillation energy flow); (B) cross-subcutaneous (the electrodes were fixed in the subcutaneous tissue of the right shoulder and the left lower chest, and the vector of the electrodes was roughly perpendicular to the direction of the energy flow); (C) parallel-surface (the electrodes were fixed with ECG paste on the shaved skin surface at the left shoulder and the right lower chest); and (D) surface grounded (the electrodes were fixed on the shaved skin surface at the left shoulder and the left foot, which was grounded). The circuit resistance was set at a variable level (100-5,000 ohms) in accordance with the resistance measured through each canine body. Leakage energies were measured in 750 defibrillation shocks with each circuit resistance in 5 dogs. The leakage energy increased in accordance with the increase of the delivered energy and the decrease of the circuit resistance in all 4 recording positions. When the circuit resistance was set at 1,000 ohms, the leakage energy during shock delivery at 34 J was 32+/-17 mJ at position A, 5+/-9 mJ at B, 10+/-9 mJ at C, and 4+/-3 mJ at D (p=0.042). The peak current was highest at position A and was 87+/-22 mA with a circuit resistance of 1,000 ohms. The power of the leakage energy depended on the delivered energy and the impedance between the electrodes. The angle between the alignment of the recording electrodes and the direction of the energy flow was another important factor in determining the leakage energy. Although the peak current of the leakage energy reached the level of macro shock, the highest leakage energy from the body surface was considerably less because of the short duration of the shock delivery.

Bezafibrate increases prebeta 1-HDL at the expense of HDL2b in hypertriglyceridemia.

Prebeta1-high density lipoprotein (prebeta1-HDL), the initial acceptor of cell-derived cholesterol, can be generated from HDL(2) by hepatic lipase. Because bezafibrate elevates lipase activity, it may increase prebeta1-HDL at the expense of HDL(2). To answer this question, we determined the apolipoprotein A-I (apoA-I) distribution in 20 hypertriglyceridemics (triglycerides>2.26 mmol/L) and 20 sex-matched normolipidemics by native 2-dimensional gel electrophoresis. At baseline, prebeta1-HDL was 70% higher in hypertriglyceridemics than in normolipidemics (123.5+/-49.9 versus 72.5+/-34.1 mg/L apoA-I, P<0.01). Prebeta1-HDL was positively correlated with triglyceride (r=0.624, P<0.0001). A 4-week bezafibrate treatment (400 mg daily) increased prebeta1-HDL by 30% (160.2+/-64.5 mg/L apoA-I, P<0.05) but decreased HDL(2b) by 31% (from 188.8+/-94.9 to 129.3+/-78.7 mg/L apoA-I, P<0.05). Hepatic lipase activity increased by 24% (P<0.005). Prebeta1-HDL was generated either from ultracentrifugally isolated HDL(2) or from plasma during incubation with triglyceride lipase. In conclusion, bezafibrate increases prebeta1-HDL at the expense of HDL(2). We speculate that such an effect might partly contribute to the antiatherogenic action of bezafibrate.

Sucrose-diet feeding induces gene expression of heat shock protein in rat brain under stress.

Stress-induced hyperphagia is enhanced in the presence of sweets, particularly sucrose, which may act to attenuate stress. Recently, it was also reported that heat shock protein (HSP) may be involved in the defense against stress. To explore whether sucrose alters gene expression of HSP under stress, we determined the HSP mRNA levels in the hypothalamus, cerebellum, and cerebral cortex after restraint stress in sucrose-diet-fed rats. Competitive RT-PCR revealed that gene expressions of HSP27 in the cerebral cortex and cerebellum and of HSP70 in the cerebral cortex, hypothalamus, and cerebellum were induced by restraint stress under a sucrose-diet-fed condition. However, restraint stress by itself or sucrose diet alone did not induce expression of HSP27 or HSP70 mRNA in any of the three anatomical parts. It is suggested that sucrose facilitates the gene expression of HSP27 and HSP70 in brain after restraint stress, which may attenuate stress.

Prebeta1-high-density lipoprotein (prebeta1-HDL) concentration can change with low-density lipoprotein-cholesterol (LDL-C) concentration independent of cholesteryl ester transfer protein (CETP).

To clarify whether prebeta1-high-density lipoprotein (prebeta1-HDL) concentration changes with low-density lipoprotein-cholesterol (LDL-C) concentration independent of cholesteryl ester transfer protein (CETP), we determined prebeta1-HDL concentration by native two-dimensional gel electrophoresis in 58 subjects with normal triglyceride and HDL-cholesterol concentrations. We also measured LDL-C and CETP concentrations. In 17 subjects, a second blood sample was taken 1-6 months after the first. We found that prebeta1-HDL concentration was positively correlated with LDL-C concentration (r=0.529, P<0.0001) and with CETP mass (r=0.398, P<0.01). In 17 patients, Deltaprebeta1-HDL was positively correlated with DeltaLDL-C (r=0.635, P<0.01), but not with DeltaCETP mass (r=0.275). In conclusion, prebeta1-HDL concentration changes with LDL-C concentration independent of CETP. These results suggest that prebeta1-HDL concentration may reflect the balance between several regulatory factors, including LDL-C and CETP concentrations.

Epitope mapping of rat neutralizing monoclonal antibody against human immunodeficiency virus type-1 by a phage peptide library: comparison with ELISA using synthetic peptides.

We generated a rat monoclonal antibody (mAb W#10) with the ability to neutralize human immunodeficiency virus type 1IIIB (HIV-1IIIB) infection. The epitope recognized by mAb W#10 was defined as R-I-Q-R-G-P-G by enzyme-linked immunosorbent assay (ELISA) with the use of synthetic peptides. The filamentous phage clones displaying random 15-amino-acid peptides on the amino terminus of the pIII coat protein reacting with mAb W#10 were identified with affinity and immunological selection procedures. Thirteen out of 16 selected phage clones contained the G-X-G-R-X-F sequence in the coat protein region representing significant homology to a part of conserved G-P-G-R-A-F sequence in the V3 loop of various HIV-1 strains. In addition, the phage clones included the G-X-G sequence in the sequence detected by synthetic peptides as the recognition site. The selected phage clones were stained by mAb W#10 specifically and were able to compete with mAb binding to cells expressing viral antigens.

Increased uncoupling protein-2 and -3 gene expressions in skeletal muscle of STZ-induced diabetic rats.

Streptozotocin (STZ)-induced diabetic animals are vulnerable to cold stress. Uncoupling proteins (UCPs) play an important role in regulating thermogenesis. We investigated the gene expressions of UCPs in brown adipose tissue (BAT), white adipose tissue (WAT), liver and gastrocnemius muscle of STZ-diabetic rats using Northern blot. UCP-1, -2 and -3 mRNA expressions in BAT were all remarkably lower in STZ-diabetic rats than those in control rats. Both UCP-2 and -3 gene expressions in gastrocnemius muscle were substantially elevated in STZ-diabetic rats and insulin treatment restored UCP gene expressions to normal levels. These results suggest that in STZ-diabetic rats, the overexpression of UCP-2 and UCP-3 in skeletal muscle provides a defense against hypothermogenesis caused by decreased UCPs in BAT.

Lipoprotein abnormalities in type 2 diabetic patients. Increased distribution of triglycerides (TG) in very low density lipoprotein (VLDL) and decreased apolipoprotein E/VLDL-TG ratio are risk for ischemic heart disease.

Plasma lipoproteins and clinical characteristics of type 2 diabetic patients with (n = 50) and without (n = 108) ischemic heart disease (IHD) were compared. The patients with IHD were older (64 +/- 9 vs 59 +/- 9 years, mean +/- SD, p < 0.01) and had a longer duration of diabetes (14 +/- 9 vs 11 +/- 8 years, p < 0.05). Lipids, lipoproteins and apolipoproteins were comparable in the two groups. The percent distribution of triglycerides (TG) in the very low density lipoprotein (VLDL) fraction was significantly higher (55 +/- 16 vs 50 +/- 17%, p < 0.05). The level of low density lipoprotein (LDL) (33 +/- 14 vs 39 +/- 15%, p < 0.05) and the apo E/VLDL-TG ratio (0.085 +/- 0.045 vs 0.120 +/- 0.097, p < 0.01), however, was significantly lower in patients with IHD than in those without IHD. Multiple regression analysis also indicated that age, duration of diabetes, percent distribution of TG in VLDL, percent distribution of TG in LDL and the apo E/VLDL-TG ratio were significantly related to the presence of IHD. Hypertriglyceridemia, particularly when characterized by abnormalities of TG distribution, may play an important role in the development of IHD in type 2 diabetic patients.