Chicken PRDX3 is required for proliferation of chicken embryo fibroblast cells.

1. This experiment investigated the influence of chicken PRDX3 on cell proliferation in chick embryo fibroblast cells using PRDX3 knockdown technology.2. A methyl thiazolyl tetrazolium (MTT) assay was performed to assess the effect of chPRDX3 knockdown on fibroblast proliferation. The antioxidant effect was investigated to determine if it directly mediated fibroblast cell proliferation.3. To determine the role of chPRDX3 on cell proliferation, an siRNA mediated knockdown was performed in chick fibroblast cells using an in vitro assay. The proliferation of fibroblast cells transfected with siPRDX3 #3 and siPRDX3 Mix was significantly decreased after 48 h (P < 0.01). In addition, the knockdown of chicken PRDX3 suppressed cell proliferation through an increase in oxidative stress.4. The results demonstrated that chPRDX3 is required for cell proliferation in chicken fibroblast cells. Such findings have important implications for the maintenance of chicken fibroblast cells.

Identification of lactoferrin and glutamate receptor-interacting protein 1 in bovine cervical mucus: A putative marker for oestrous detection.

Accurate detection of oestrus is important for artificial insemination. The aim of this study was to identify oestrous-specific bovine cervical mucus proteins that could be used to determine the optimal time for artificial insemination. Non-oestrous and controlled internal drug release (CIDR)-induced oestrous-stage mucus proteins were purified and subjected to surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, sodium dodecyl sulphate polyacrylamide gel electrophoresis and MALDI-TOF/TOF. Among differentially expressed proteins, lactoferrin (LF) and glutamate receptor-interacting protein 1 (GRIP1) showed a twofold increase during the CIDR-induced oestrous stage compared to the levels in non-oestrous stage in bovine cervical mucus. The RT-PCR, Western blotting and immunohistochemistry results showed that LF and GRIP1 expression was significantly increased during the oestrous stage in the uterus. This study demonstrated that bovine LF and GRIP1 exist during the oestrous stage, but not during the non-oestrous stage, suggesting that cervical mucus LF and GRIP1 are useful oestrous detection markers in cattle.

Tsg101 is upregulated in a subset of invasive human breast cancers and its targeted overexpression in transgenic mice reveals weak oncogenic properties for mammary cancer initiation.

Previous studies reported that the Tumor Susceptibility Gene 101 (TSG101) is upregulated in selected human malignancies, and the expression of exogenous Tsg101 was suggested to transform immortalized fibroblasts in culture. To date, the potential oncogenic properties of Tsg101 have not been examined in vivo owing to the lack of appropriate model systems. In this study, we show that Tsg101 is highly expressed in a subset of invasive human breast cancers. Based on this observation, we generated the first transgenic mouse model with a targeted overexpression of Tsg101 in the developing mammary gland to test whether exogenous Tsg101 is capable of initiating tumorigenesis. Normal functionality of exogenous Tsg101 was tested by rescuing the survival of Tsg101-deficient mammary epithelial cells in conditional knockout mice. The overexpression of Tsg101 resulted in increased phosphorylation of the epidermal growth factor receptor and downstream activation of MAP kinases. Despite an increase in the activation of these signal transducers, the mammary gland of females expressing exogenous Tsg101 developed normally throughout the reproductive cycle. In aging females, the overexpression of Tsg101 seemed to increase the susceptibility of mammary epithelia toward malignant transformation. However, owing to the long latency of tumor formation and the sporadic occurrence of bona fide mammary cancers, we conclude that the Tsg101 protein has only weak oncogenic properties. Instead of cancer initiation, it is therefore likely that Tsg101 plays a more predominant role in the progression of a subset of spontaneously arising breast cancers.

Effects of dietary recombinant chlorella supplementation on growth performance, meat quality, blood characteristics, excreta microflora, and nutrient digestibility in broilers.

The use of chlorella as an immune stimulant to enhance nonspecific host defense mechanisms or as an antimicrobial to inhibit bacterial growth has been reported. Thus, the aim of the present study was to clarify the effect of recombinant chlorella supplementation on growth performance, meat quality, and the blood profile, excreta microflora, and nutrient digestibility in broilers. A total of 375 one-day-old ROSS 308 broilers (male and female) were allotted to 5 dietary treatments using 5 cages with 15 chicks per cage. Treatments were: 1) NC, basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) PC1, 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) T1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). The broilers in the T2 treatment groups showed higher body weight gain (BGW) by 2.55% (P < 0.01) and lower feed conversion ratio (FCR) by 2.75% (P < 0.05) compared with those fed the control NC treatment group. Moreover, the blood contents of blood urea nitrogen (BUN), creatinine, and IgA in the broilers of the T2 treatment group were significantly increased by 28.12, 23.07, and 29.72%, respectively -more than those found in the broilers of the NC treatment group (P < 0.01). In contrast, the LDL/C in the blood from the animals in the T2 treatment group was significantly decreased by 23.23% - more than that in the blood from the NC broilers (P < 0.05). Based on these results, we suggest that the dietary supplementation of broilers with recombinant chlorella could improve their growth performance, increase the concentration of IgA and apparently metabolizable nitrogen in the blood, and decrease ammonia emissions. Therefore, our findings have important implications for the effect of recombinant chlorella supplementation through increasing the concentration of IgA and the level of metabolizable nitrogen.

A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli.

A novel fluorescent derivative of glucose was synthesized by reacting D-glucosamine and NBD-Cl. The TLC analysis of the reaction mixture showed the generation of a single spot with intense fluorescence (lambda Ex = 475 nm, lambda Em = 550 nm). The obtained novel fluorescent product, which was identified as 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) by 1H-NMR and FAB-MS spectrometries, was applied to the assessment of the glucose uptake activity of Escherichia coli B. 2-NBDG accumulated in living cells and not in dead cells. The uptake of 2-NBDG was competitively inhibited by D-glucose and not by L-glucose, which suggested the involvement of the glucose transporting system in the uptake of 2-NBDG. 2-NBDG taken into the cytoplasma of E. coli cells was supposedly converted into another derivative in the glucose metabolic pathway.

Flow sensing in mycelial fungi.

Hyphae of a variety of mycelial fungi were shown to reorient their direction of growth to grow upstream in a continuous flowing current of liquid medium. Among those tested fungi, Aspergillus niger hyphae was most polarized towards the upstream. Hyphae of A. niger which had been attached to poly-L-lysine coated plates on the base of a continuous-culture flow cell were exposed to a current of medium of flow rates in the range of 0-6400 microns s-1 for 1-4 h. Hyphal tips quickly reoriented towards the upstream of the flowing medium and became aligned parallel to the flow axis as they grew larger. At flow rates above 1600 microns s-1, however, the percentage of polarization of hyphae reached a constant level. With other fungi, germ tube emergence was not polarized but hyphae and branches grew towards the upstream. There was no detectable difference in the pH and oxygen concentration of the flowing medium between inlet and outlet of the flow cell. This result suggests that hyphae of mycelial fungi sense transient flow of liquid medium over their surface and reorient their direction to grow upstream.

Detection of antifungal activity in Anemarrhena asphodeloides by sensitive BCT method and isolation of its active compound.

Antifungal activity was detected from Anemarrhena asphodeloides by the Bio-Cell Tracer (BCT) method. An active fraction was separated by silica gel column chromatography and reverse-phase HPLC. The molecular weight was determined by GC-MS, and the molecular structure was analyzed by IR, (1)H NMR, and (13)C NMR. The isolated compound was found to be identical to nyasol, (Z)-1, 3-bis(4-hydroxyphenyl)-1,4-pentadiene, which formerly appeared in the literature without any remark on the antifungal activity. This compound showed antimicrobial activity against 38 strains of fungi and five strains of bacteria. The minimum inhibitory concentration (MIC) ranged from 12.5 to 200 microg mL(-)(1), except for two strains based on the broth dilution method.

Intraoperative assessment of hepatic venous congestion with direct clamping of the hepatic vein trunk for living donor liver transplantation.

We devised a hepatic vein clamping method to assess the amount of hepatic venous congestion (HVC) before liver transection. From February 2003 to May 2003, this method was applied to 5 of 58 living donor livers especially to assess donor safety. The left portal vein and proper hepatic artery as well as the middle hepatic vein (MHV)-left hepatic vein (LHV) trunk were clamped simultaneously to assess the HVC in the remnant right lobe before performing extended left lobectomy. As three donors demonstrated the extent of the HVC equivalent to about 40% of the right lobe volume (RLV), their operations proceeded according to the preoperative plan. The territory of HVC after liver transection was the same as that observed with direct clamping of the hepatic vein. However, one donor showed massive HVC more than 50% of RLV and the operative plan was adjusted to harvest only the left lobe without the MHV trunk for donor safety. To assess the HVC in the remnant left lobe, the isolated LHV trunk was occluded after clamping the donor's proper hepatic artery. The whole left lobe except for a small area at the anterior portion of the medial segment became discolored on LHV clamping: the opposite demarcation appeared on MHV clamping. The amount of HVC was so small that we harvested the right lobe with the MHV trunk. All donors and recipients recovered uneventfully. We believe that this direct clamping method makes the assessment of HVC feasible before parenchymal transection of a donor liver.

Evaluation of antifungal activity of an antifungal drug by in vitro simulation of in vivo pharmacokinetics of the drug against fungal hyphal growth.

An automatic drug concentration simulator (DCS) has been developed and its applicability has been demonstrated by in vitro simulation of the human plasma concentration-time curve of fluconazole (FLCZ) against hyphal growth of Candida albicans and Aspergillus fumigatus. The response of hyphal growth to FLCZ was continually monitored and analyzed using an automatic hyphal growth analyzing system (Bio-Cell Tracer). The simulated concentration of FLCZ by DCS was confirmed by HPLC. The DCS assay was reproducible with a mean coefficient of variation (C.V., n=3) of 5.38 %. When the growth of C. albicans hyphae was tested, there was a lag of onset of FLCZ effect between the time when FLCZ concentration became maximal (C MAX, 7.95 microg/ml ) and the point at which hyphal growth ceased. In contrast, FLCZ was found inactive against A. fumigatus. The newly devised technique could provide clinicians with important information in determining optimal dosing regimens for antifungal drugs.

[Sensitive screening of antifungal compounds from acetone extracts of medicinal plants with a Bio-Cell Tracer].

Antifungal activity of medicinal plants against Aspergillus niger was evaluated using Bio-Cell Tracer (BCT). By this system enabled the real time determination of hyphal growth rate could be done in the presence or absence of Chinese herbal extracts. Of the 41 herbal extracts with 60% acetone extracts tested by the present method, 26 were found to contain active components against A. niger hyphal growth. In contrast, by conventional methods, no active component was detected from every herbal extract except 3 herbal extracts. From the extract of Anemarrhena asphodeloides, one of active components was isolated and its structure determined by NMR, UV, and mass spectroscopy. The compound was identified as broussonin A (2-3-(4-hydroxyphenyl)propyl)-5-methoxy-phenol) which was formerly reported as a phytoalexin of Broussonetia papyrifera Vent.

Rapid and sensitive screening of antifungal activity in medicinal plants by a single-cell biosensing system.

A biosensing system based on the response of fungal cells was used for the evaluation of antifungal activity of medicinal plants against Aspergillus niger. This system measured the hyphal growth rate in real time in the presence or absence of Chinese herbal extracts. The sensitivity of this system was 100-fold higher than that of conventional methods, and is advisable for the screening of antifungal compounds.

Purification and characterization of an autoregulatory substance capable of regulating the morphological transition in Candida albicans.

The yeast Candida albicans has a distinguishing feature, dimorphism, which is the ability to switch between two morphological forms: a budding yeast form and a multicellular invasive filamentous form. This ability has been postulated to contribute to the virulence of this organism. Studies on the morphological transition from a filamentous to a budding yeast form in C. albicans have shown that this organism excretes an autoregulatory substance into the culture medium. This substance was extracted and purified by normal-phase and reversed-phase HPLC. The autoregulatory substance was structurally identified as 3,7,11-trimethyl-2,6,10-dodecatrienoate (farnesoic acid) by NMR and mass spectrometry. Growth experiments suggest that this substance does not inhibit yeast cell growth but inhibits filamentous growth. These findings have implications for developmental signaling by the fungus and might have medicinal value in the development of antifungal therapies.

Detection of antifungal activity in Belamcanda chinensis by a single-cell bioassay method and isolation of its active compound, tectorigenin.

The antifungal activity of Belamcanda chinensis was evaluated by a single-cell bioassay method. An active fraction was separated by silica gel column chromatography and reverse-phase HPLC. The isolated compound was found to be identical to tectorigenin (5,7-dihydroxy-3-(4-hydroxy phenyl)-6-methoxy-4H-1-benzopyran-4-one) which has formerly appeared in the literature without any remarks on its antimicrobial activity. Antimicrobial activity was investigated against 17 strains of fungi and 6 strains of bacteria. This compound showed marked antifungal activity against dermatophytes of the genera Trichophyton, the minimum inhibitory concentration (MIC) being in the range of 3.12-6.25 mg/ml.

In vitro synergism between nyasol, an active compound isolated from Anemarrhena asphodeloides, and azole agents against Candida albicans.

The antifungal activity of nyasol (NYS) alone or with various antifungal agents was measured in vitro against Candida albicans, Aspergillus fumigatus, and Trichophyton mentagrophytes. NYS is a compound recently purified from a medicinal plant, Anemarrhena asphodeloides. Among 12 agents, miconazole (MCZ), ketoconazole (KCZ), clotrimazole (CTZ), and cerulenin showed marked synergistic effects against C. albicans. The fractional inhibition concentration (FIC) indices against 4 strains of C. albicans were 0.067-0.31 for MCZ plus NYS, 0.078-0.31 for KCZ plus NYS, and 0.098-0.13 for CTZ plus NYS. These values indicate the possibility of using NYS as an adjuvant to azole agents in the chemotherapy of candidiasis.

Detection of antifungal activity in Portulaca oleracea by a single-cell bioassay system.

The antifungal activity of Portulaca oleracea extracts against hyphal growth of various fungi was evaluated in real time using an automatic single-cell bioassay system. Target organisms were the filamentous fungi Aspergillus and Trichophyton and the yeast Candida. A colony of test fungi was in contact with the assay medium, or assay medium containing plant extract, in sequence. The antifungal activity of each fraction of P. oleracea was evaluated based on the dynamic hyphal growth response curves of test fungi. A crude sample obtained by EtOAc extract showed a specific and marked activity against dermatophytes of the genera Trichophyton.

Combined effect of amphotericin B and flucytosine on hyphal growth of Candida albicans estimated at a single hypha level.

The in vitro efficacy of amphotericin B and flucytosine, separately and in combination, against the hyphal growth of Candida albicans was evaluated in situ using an automatic analysing system. A colony of C. albicans was in contact with a glucose-salt medium supplemented with biotin plus calf serum (GS medium) and GS medium containing the antifungal agent, in sequence. Minimum inhibitory concentrations at single hyphal level (S-MIC) were determined based on the response that was measured. Amphotericin B S-MICs ranged from 0.8 to 0.4 microgram ml-1, and S-MIC values > 102.4 micrograms ml-1 were obtained with flucytosine when the two agents were used independently. When the two agents were used in combination, however, a synergistic interaction between the two agents at concentrations below their individual S-MICs was observed.

Transmission of the effect of an antifungal agent within a single hypha.

A micro-compartment culture method was devised in which a single hypha of Rhizopus stolonifer growing on an agar section traversed an antifungal non-diffusible barrier to another agar section; thus the local environment of the distal or proximal part of the hypha could be controlled independently. The responses in terms of hyphal extension of the test fungus to local application of amphotericin B in this culture system were estimated by using an automatic analysing system. After hyphae had traversed the barrier, distal application of amphotericin B caused no appreciable effect on the proximal hyphae. In contrast, proximal application of amphotericin B caused inhibition of the extension of distal hyphae. The reversal of polarized cytoplasmic streaming also occurred during the inhibition of distal hyphal extension. The extents of inhibition of the distal hyphal extension and the cytoplasmic streaming were dependent upon the hyphal distance between the amphotericin B application site and the hyphal tip. These results show that the effect of an antifungal agent on a hypha depends on the region of the hypha exposed. Cytoplasmic streaming may play key role in the transmission of antifungal effects within a single hypha.

Evaluation of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose, a new fluorescent derivative of glucose, for viability assessment of yeast Candida albicans.

A new fluorescent derivative of D-glucose, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), which had been previously developed for the analysis of glucose uptake activity by living cells, was investigated to evaluate its applicability for assaying the viability of yeast Candida albicans. Lineweaver-Burk plots showed to uptake of 2-NBDG to be competitively inhibited by D-glucose and not by L-glucose, which suggested the involvement of the glucose transporting system of C. albicans in the uptake of 2-NBDG. A good correlation was obtained between the yeast viability, determined by the plate-count method, and the 2-NBDG uptake activity of yeast cells (correlation constant: r = 0.97). This is expected to lead to the development of a new fluorescent probe for the determination of yeast cell viability.

Effects of a pulsing electric signal on the cross membrane potential and the cell division potentiality of a single cell of tobacco.

A pulsing electric signal (pulse width 10 s) was applied to a single cell of cultured tobacco, line BY-2, by inserting a multifunctional microelectrode (MME) into the cell. The electric voltage (V(ET)) was loaded between the electrode terminals of the MME and the reference electrode situated in the extracellular medium. Since the electrical impedance of the MME was as large as that of the cell membrane, the effective potential acting across the cell membrane (V(CMP)) should be only some portion of V(ET). The MME enabled simultaneous measurement of V(ET) and V(CMP). When V(ET) was varied from 0 to -1 V, V(CMP) changed linearly in proportion to V(ET). When V(ET) variation range was enlarged (from 0 to -2 V), V(CMP) changing pattern became a declined curve. When V(ET) variation range was further enlarged (from 0 to -5 V), the V(CMP) changing pattern showed a saturation curve. Under this condition, the cell division potentiality decreased accordingly. Based on these results, the feasibility of V(CMP) as an indicator of the effective intensity of an electric stress signal is discussed. In the present case of a BY-2 cell, a proper intensity of V(CMP) that could cause an appreciable stress and not a lethal signal was estimated as -250 mV.