A fluorescent antibiotic resistance marker for rapid production of transgenic rice plants

Blasticidin S (BS) is an aminoacylnucleoside antibiotic used for the control of rice blast disease. To establish a new cereal transformation system, we constructed a visual marker gene designated gfbsd, encoding an enhanced green fluorescent protein (EGFP) fused to the N-terminus of BS deaminase (BSD). It was cloned into a monocot expression vector and introduced into rice (Oryza sativa L. cv. Nipponbare) calluses by microprojectile bombardment. Three to five weeks after the bombardment, multicellular clusters emitting bright-green EGFP fluorescence were obtained with 10 microg/ml BS, which is not sufficient to completely inhibit the growth of non-transformed tissues. Fluorescent sectors (approximately 2mm in diameter) excised from the calluses regenerated into transgenic plantlets (approximately 10 cm in height) as early as 51 (average 77+/-11) days after the bombardment. The visual antibiotic selection was more efficient and required less time than the bialaphos selection with bar. In addition, the small size (1.1 kb) of gfbsd is preferable for construction of transformation vectors. This new marker gene will make a significant contribution in molecular genetic studies of rice plants.     

Nutrition of captive lowland anoa (Bubalus depressicornis): a study on ingesta passage,intake, digestibility,and a diet survey

Members of the Bovini genus are classified as grazers. Smaller species of ruminants are not expected to be able to digest particularly fibrous diets and are more often classified as intermediate feeders or browsers. Anoas (Bubalus spp.) are interesting in this respect as they are the smallest representatives of the Bovini, being only 10–20% of the body weight of other species of the same genus. A feeding trial was carried out with four lowland anoas (Bubalus depressicornis) at London Zoo, investigating diet digestibility by total fecal collection and passage rates by the simultaneous administration of a fluid (Co‐EDTA) and a particle (Cr‐mordanted fibre <2 mm) marker. The diet consisted of legume hay, dairy cow pellets, browse, fruits, and vegetables. The achieved digestibility coefficients averaged 70±4% for dry matter and 57±7% for cell walls (NDF). Mean retention times for the total gastrointestinal tract were 25±4.1 hr for fluid and 39±6.7 hr for particles, respectively. The ratio of forestomach particle:fluid retention was 2.14±0.40. Additional information regarding anoa diets in captivity was collected through a survey targeting all institutions that have anoas in their collection currently. Suitability of the provided diet was evaluated using the ratio of unstructured:structured feeds (unstructured feeds pellets, grains, produce; structured feeds=roughage, browse) on a dry matter basis and an assumed complete consumption of offered unstructured diet items, with only the remaining intake capacity being met by structured items. The use of this ratio reliably predicted one facility that reported chronic diet‐related problems. As other ruminants, anoas should receive a diet with restricted amounts of concentrates and fruits. The comparatively high fibre digestibility and the high selective particle retention in the forestomach suggest a classification of an intermediate/grazing ruminant. Zoo Biol 24:125–134, 2005. © 2005 Wiley‐Liss, Inc.     

Anthracene based base-discriminating fluorescent oligonucleotide probes for SNPs typing: synthesis and photophysical properties

2- and 9-Anthracenecarboxamide labeled 2'-deoxyuridines were synthesized and their photophysical properties were examined. These oligonucleonucleotide probes are capable of detecting adenine base on a target DNA sequence. It was also found that 2-anthracene based oligonucleotide probe is more efficient than the corresponding 9-anthracene based oligonucleotide in the application for DNA chip based SNP detection, due to its longer emission wavelength and high fluorescence intensity.     

Translocation of VP1686 upregulates RhoB and accelerates phagocytic activity of macrophage through actin remodeling

Here, we report that Vibrio parahaemolyticus induces a rapid remodeling of macrophage actin and activates RhoB GTPase. Mutational analysis revealed that the effects depend on type III secretion system 1 regulated translocation of a V. parahaemolyticus effector protein, VP1686, into the macrophages. Remodeling of actin is shown to be necessary for increased bacterial uptake followed by initiation of apoptosis in macrophages. This provides evidence for functional association of the VP1686 in triggering an eat-me-and-die signal to the host.     

Live rats resulting from injection of oocytes with spermatozoa freeze-dried and stored for one year

This study was designed to examine whether rat spermatozoa after freeze-drying and 1-year storage can participate in full-term development following intracytoplasmic sperm injection (ICSI). Cauda epididymal spermatozoa from Crlj:Wistar rats were frozen in liquid nitrogen (LN(2)), first dried for 14 hr at 0.37 hPa and then for 3 hr at 0.001 hPa. The dried spermatozoa were stored for 1 year in a desiccator at +25 degrees C, or in a refrigerator at +4 degrees C, or in LN(2) at -196 degrees C. Controls consisted of sperm that had only been frozen and stored in LN(2). After being stored, spermatozoa were sonicated to dissociate the sperm tail and were injected into oocytes from superovulated Slc:SD rats. The respective fertilization rates of oocytes injected with frozen sperm, or with freeze-dried sperm stored at +25, +4, and -196 degrees C were 79%, 75%, 70%, and 73%. However, the corresponding cleavage rates of injected oocytes were 63%, 1%, 38%, and 36%. After transfer of >80 zygotes of each group into recipients, the respective percentages of full-term normal offspring resulting from frozen sperm or from freeze-dried sperm stored at +25, +4, and -196 degrees C were 36%, 0%, 7%, and 14%. These results demonstrate that the storage temperature significantly influenced the likelihood of term development of rats produced by injection of oocytes with freeze-dried spermatozoa. Chromosomal analysis of the rat spermatozoa in the ICSI oocytes indicated that chromosomal aberration in freeze-dried spermatozoa stored at +25 degrees C (100%) occurred more frequently than in frozen control spermatozoa (41%) and freeze-dried spermatozoa stored at -196 degrees C (35%), and the frequency of chromosomal aberrations in freeze-dried spermatozoa stored at +4 degrees C (65%) was the intermediate. In conclusion, rat spermatozoa freeze-dried and stored at +4 degrees C for 1 year are capable of participating in full-term development after ICSI.     

High-pressure study on bilayer phase behavior of oleoylmyristoyl- and myristoyloleoyl-phosphatidylcholines

We investigated the thermotropic and barotropic bilayer phase behavior of 1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC) and 1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (OMPC) by means of the differential scanning calorimetry (DSC) and high-pressure light-transmittance technique. Water could be used as a solvent for measurements at high pressures because of the elevation of the transition temperatures above 0 degrees C by pressurization, whereas aqueous 50 wt.% ethylene glycol solution was used mainly for those at low pressures. Only one phase transition was observed in the DSC thermogram of the MOPC bilayer membrane as an endothermic peak, and also observed at high pressures as an abrupt change of the light-transmittance. The transition was assigned as a main transition between the lamellar gel (L(beta)) and liquid-crystalline (L(alpha)) phases on the basis of the values of enthalpy change (DeltaH) and slope of the transition temperature with respect to pressure (dT/dP). The DSC thermogram of the OMPC bilayer membrane similarly showed a single endothermic peak but two kinds of phase transitions were observed at different temperatures in the light-transmittance profile at high pressures. The extrapolation of the lower-temperature transition in the high-pressure range to an ambient pressure coincided with the transition observed in the DSC thermogram. This transition was identified as a transition between the lamellar crystal (L(c)) and L(alpha) (or L(beta)) phases from the DeltaH and dT/dP values. The higher-temperature transition, appearing only at high pressures, was identified as the L(beta)/L(alpha) transition considering the topological resemblance of its temperature-pressure phase diagram as that of the dioleoylphosphatidylcholine bilayer membrane. The phase diagram of the OMPC bilayer membrane demonstrated that the L(beta) phase cannot exist at pressures below ca. 190 MPa while it can exist stably in a finite temperature range at pressures above the pressure.     

Role of seed coat in imbibing soybean seeds observed by micro-magnetic resonance imaging

Background and Aims

Imbibition of Japanese soybean (Glycine max) cultivars was studied using micro-magnetic resonance imaging (MRI) in order to elucidate the mechanism of soaking injury and the protective role of the seed coat.

Methods

Time-lapse images during water uptake were acquired by the single-point imaging (SPI) method at 15-min intervals, for 20 h in the dry seed with seed coat, and for 2 h in seeds with the seed coat removed. The technique visualized water migration within the testa and demonstrated the distortion associated with cotyledon swelling during the very early stages of water uptake.

Key Results

Water soon appeared in the testa and went around the dorsal surface of the seed from near the raphe, then migrated to the hilum region. An obvious protrusion was noted when water reached the hypocotyl and the radicle, followed by swelling of the cotyledons. A convex area was observed around the raphe with the enlargement of the seed. Water was always incorporated into the cotyledons from the abaxial surfaces, leading to swelling and generating a large air space between the adaxial surfaces. Water uptake greatly slowed, and the internal structures, veins and oil-accumulating tissues in the cotyledons developed after the seed stopped expanding. When the testa was removed from the dry seeds before imbibition, the cotyledons were severely damaged within 1·5 h of water uptake.

Conclusions

The activation of the water channel seemed unnecessary for water entry into soybean seeds, and the testa rapidly swelled with steeping in water. However, the testa did not regulate the water incorporation in itself, but rather the rate at which water encountered the hypocotyl, the radicle, and the cotyledons through the inner layer of the seed coat, and thus prevented the destruction of the seed tissues at the beginning of imbibition.Key words: Dry seeds, Glycine max, MRI, seed coat, soaking injury, soybean, testa, role of inner layer of seed coat, water uptake     

Flavonoids in the leaves of <Emphasis Type="Italic">Oxalis corniculata </Emphasis>and sequestration of the flavonoids in the wing scales of the pale grass blue butterfly, <Emphasis Type="Italic">Pseudozizeeria </Emphasis><Emphasis Type="Italic">maha</Emphasis>

Three C-glycosylflavones in the leaves of Oxalis corniculata, the host plant of the lycaenid butterfly pale grass blue (Pseudozizeeria maha), were identified as 6-C-glucosylluteolin (isoorientin), 6-C-glucosylapigenin (isovitexin) and isovitexin 7-methyl ether (swertisin). Comparative spectral and HPLC analyses between the leaf extract of the host plants and the wings of P. maha showed selective uptake of the host-plant flavonoid isovitexin to the wings of the butterfly.     

Determination of amobarbital and phenobarbital in serum by gas chromatography-mass spectrometry with addition of formic acid to the solvent

A rapid and accurate method for quantification of amobarbital and phenobarbital was developed using gas chromatography-mass spectrometry (GC-MS) without derivatization. Though the compounds measured without derivatization showed low sensitivity because of adsorption, addition of 3% formic acid to the solvent improved the sensitivity for the analytes. Taking account of matrix effect, solid-phase and liquid-liquid extraction from serum were examined. The correlation coefficients of the calibration curves were 0.9995 or better, and the accuracy and precision of intraday and interday assays were in line with Food and Drug Administration (FDA) criteria.     

Release of cytokines/chemokines and cell death in UVB-irradiated human keratinocytes, HaCaT

Ultraviolet (UV) B can lead to inflammatory responses such as sunburn, which involves the production of various inflammatory cytokines and chemokines, and the induction of cell death. Keratinocytes in the skin has one of the highest risks of exposure to UV. However, the detailed mechanisms underlying UVB irradiation-induced inflammation and cell death are not well known. Thus, we investigated the effect of UVB irradiation on the production of various cytokines/chemokines and the induction of cell death in UVB-irradiated human keratinocytes (HaCaT cells). We evaluated 11 cytokines/chemokines in cell culture supernatants from HaCaT cells exposed to 0-400 mJ/cm(2) UVB irradiation. UVB at a dose 400 mJ/cm(2) induced the release of various cytokines; interleukin (IL)-1beta, IL-6, IL-8, interferon (IFN)-gamma, granulocyte-colony stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, and tumor necrosis factor (TNF)-alpha. These results suggest that UVB irradiation-induced the release of several cytokines/chemokines and led to cell death in human keratinocytes. UV exposure may be associated with multiple physiological events in the human skin.