Optimization of a transplant model to assess skin reconstitution from stem cell-enriched primary human keratinocyte populations

Given that an important functional attribute of stem cells in vivo is their ability to sustain tissue regeneration, we set out to establish a simple and easy technique to assess this property from candidate populations of human keratinocyte stem cells in an in vivo setting. Keratinocytes were inoculated into devitalized rat tracheas and transplanted subcutaneously into SCID mice, and the epithelial lining regenerated characterized to establish the validity of this heterotypic model. Furthermore, the rate and quality of epidermal tissue reconstitution obtained from freshly isolated unfractionated vs. keratinocyte stem cell-enriched populations was tested as a function of (a) cell numbers inoculated; and (b) the inclusion of irradiated support keratinocytes and dermal cells. Rapid and sustained epidermal tissue regeneration from small numbers of freshly isolated human keratinocyte stem cells validates the utilization of this simple and reliable model system to assay for enrichment of epidermal tissue-reconstituting cells.

Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3 '-kinase-dependent machinery

In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3'-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes.

Effect of free ammonia and free nitrous acid concentration on the anabolic and catabolic processes of an enriched Nitrosomonas culture

The effects of free ammonia (FA; NH3) and free nitrous acid (FNA; HNO2) concentrations on the metabolisms of an enriched ammonia oxidizing bacteria (AOB) culture were investigated using a method allowing the decoupling of growth and energy generation processes. A lab-scale sequencing batch reactor (SBR) was operated for the enrichment of an AOB culture. Fluorescent in-situ hybridization (FISH) analysis showed that 82% of the bacterial population in the SBR bound to the NEU probe specifically designed for Nitrosomonas europaea. Batch tests were carried out to measure the oxygen and ammonium consumption rates by the culture at various FA and FNA levels, in the presence or absence of inorganic carbon (CO2, HCO3, and CO32-). It was revealed that FA of up to 16.0 mgNH(3)-N (.) L-1, which was the highest concentration used in this study, did not have any inhibitory effect on either the catabolic or anabolic processes of the Nitrosomonas culture. In contrast, FNA inhibited both the growth and energy production capabilities of the Nitrosomonas culture. The inhibition on growth initiated at approximately 0.10 mgHNO(2)-(NL-1)-L-., and the data suggested that the biosynthesis was completely stopped at an FNA concentration of 0.40 mgHNO(2)-N (.) L-1. The inhibition on energy generation initiated at a slightly lower level but the Nitrosomonas culture was still oxidizing ammonia at half of the maximum rate at an FNA concentration of 0.50-0.63 mgHNO(2)-N (.) L-1. The affinity constant of the Nitrosomonas culture with respect to ammonia was determined to be 0.36 mgNH3-N (.) L-1, independent of the presence or absence of inorganic carbon. (c) 2006 Wiley Periodicals, Inc.

Genotypic consistency in the expression of leaf water potential in rice (Oryza sativa L.)

Early work has shown variation in the grain yield of rice cultivars grown under water stress conditions to be associated with the plant water status, mainly with the maintenance of high leaf water potential (LWP) at flowering and grain filling stage. Considerable variation for LWP among rice varieties has been recorded. The present work was designed to investigate genotypic consistency in water potential within the plant and under canopy manipulation to vary plant water requirement. In a glasshouse experiment, with six rice genotypes, a consistent water potential gradient from stem base to leaf tip has been observed. Leaf tip water potential has been found as the minimum LWP that can be recorded at any time of stress. Genotypes with similar canopy size could maintain different levels of LWP under stress conditions. In a field experiment, with four selected lines, four canopy sizes and two canopy mixture treatments were introduced prior to the imposition of control, mild and severe water stress conditions. It was found that the line differences in LWP and relative water content (RWC) were expressed under both mild and severe stress conditions, regardless of canopy size, tiller number and whether they were mixed with another line with different capacity to maintain LWP. Although there were some differences among canopy size treatments for radiation interception in three water conditions, canopy manipulation (plant size) within a line did not affect the expression of LWP and hence genotypic variation in LWP was maintained. Under both glasshouse and field conditions, lines that maintained high LWP had larger xylem diameter and stem areas than those that had low LWP. The results indicated that the size of the vascular bundles could influence the maintenance of plant water relations under water deficit. (c) 2005 Elsevier B.V. All rights reserved.

Genotypic variation for cold tolerance during reproductive development in rice: Screening with cold air and cold water

Rice (Oryza sativa L.) plants are susceptible to low temperature during the young microspore stage, which occurs 10-12 days before heading. Low temperature at this time increases spikelet sterility which can cause massive yield loss. Increasing the cold tolerance of cultivars can reduce yield variability in temperate rice-growing environments. Two experiments were conducted in cold air screenings and two were conducted in cold water screenings to examine genotypic variation for cold tolerance, explore flowering traits related to spikelet sterility, and investigate whether the results reflect the level of cold tolerance determined previously in the field. Cold air screenings imposed day/night temperatures of 27 degrees C/13 degrees C, 25 degrees C/15 degrees C and 32 degrees C/25 degrees C following particle initiation until 50% heading, while cold water screenings maintained a relatively constant 19 degrees C. The variation in the commencement of low air temperature treatment did not have an effect on the level of spikelet sterility, indicating that exposure to low temperature during the young microspore stage was more important than the duration of exposure. Spikelet sterility of common cultivars showed a significant correlation between cold air and cold water screenings (r(2) = 0.63, p < 0.01), cold air and field screenings (r(2) = 0.52, p < 0.01) and cold water and field screenings (r(2) = 0.53, p < 0.01), indicating that cold air and cold water can be used for screening genotypes for low temperature tolerance. HSC55, M 103 and Jyoudeki were identified as cold tolerant and Doongara, Sasanishiki and Nipponbare as susceptible cultivars. There was a significant negative relationship between spikelet sterility and both the number of engorged pollen grains per anther and anther area only after imposing cold air and cold water treatment hence, it was concluded that these flowering traits were facultative in nature. In addition, cultivars originating from Australia and California were inefficient at producing filled grain with similar sized anthers containing a similar number of engorged pollen grains as cultivars from other origins. One suggested reason for this poor conversion to filled grain of cultivars from Australia and California may be associated with their small stigma area, particularly when exposed to low temperature conditions. (c) 2006 Elsevier B.V. All rights reserved.

Improving drought tolerance in rainfed lowland rice: An example from Thailand

A large portion of the world's poor farm in rainfed systems where the water supply is unpredictable and droughts are common. In Thailand there are approximately 6.2 million ha of rain fed lowland rice, which account for 67% of the country's total rice-growing area. This rice system is often characterised by too much and too little water in the same season. Farmers' estimates of their annual losses to drought are as high as 45% in the upper parts of the toposequence. In contrast to irrigated rice systems, gains from crop improvement of rainfed rice have been modest, in part because there has been little effort to breed and select for drought tolerance for the target rainfed environments. The crop improvement strategy being used in Thailand considers three mechanisms that influence yield in the drought prone targets: yield potential as an important mechanism for mild drought (where yield loss is less than 50%), drought escape (appropriate phenology) and drought tolerance traits of leaf water potential, sterility, flower delay and drought response index for more severe drought conditions. Genotypes are exposed to managed drought environments for selection of drought tolerant genotypes. A marker assisted selection (MAS) scheme has been developed and applied for selection of progenies in the backcrossing program. The plant breeding program uses rapid generation advance techniques that enable early yield testing in the target population of environments (TPE) through inter-station (multi-location yield testing) and on-farm trials. A farmer participatory approach has been used to identify the TPE for the breeding program. Four terrace paddy levels have been identified, upper (drought), middle (drought prone to favorable) and lower (flooded). This paper reports the change in the breeding program for the drought prone tainted lowland rice environments of North and Northeast Thailand by incorporating our knowledge on adaptation and on response of rice to drought. (c) 2005 Elsevier B.V. All rights reserved.

Iron (Fe) bioavailability and the distribution of anti-Fe nutrition biochemicals in the unpolished, and bran fraction of five rice gei polished grain riotypes

Iron (Fe) bioavailability in unpolished, polished grain and bran fraction of five rice genotypes with a range of Fe contents was measured by in vitro digestion and cultured Caco-2 cells of cooked grain. There was a significant difference in Fe bioavailability among the five rice genotypes tested, in both the unpolished and polished grain. The range of Fe bioavailability variation in polished rice was much wider than that of unpolished, suggesting the importance of using Fe levels and bioavailability in polished rice grain as the basis for selecting high-Fe rice cultivars for both agronomic and breeding purposes. Milling and polishing the grain to produce polished (or white) rice increased Fe bioavailability in all genotypes. Iron bioavailability in polished rice was high in the UBON2 and Nishiki, intermediate in both IR68144 and KDML105, and low in CMU122. All genotypes had low bioavailability of Fe in bran fraction compared to unpolished and polished grain, except in CMU122. CMU122 contained the lowest level of bioavailable Fe in unpolished and polished grain and bran, because of the dark purple pericarp colored grain and associated tannin content. The level of bioavailable Fe was not significantly correlated with grain Fe concentration or grain phytate levels among these five genotypes tested. The negative relationship between Fe bioavailability and the levels of total extractable phenol was only observed in unpolished (r = -0.83**) and bran fraction (r = -0.50*). The present results suggested that total extractable phenol and tannin contents could also contribute to lowering bioavailability of Fe in rice grain, in addition to phytate. (c) 2006 Society of Chemical Industry

Minimising cold damage during reproductive development among temperate rice genotypes. I. Avoiding low temperature with the use of appropriate sowing time and photoperiod-sensitive varieties

Multiple-sown field trials in 4 consecutive years in the Riverina region of south-eastern Australia provided 24 different combinations of temperature and day length, which enabled the development of crop phenology models. A crop model was developed for 7 cultivars from diverse origins to identify if photoperiod sensitivity is involved in determining phenological development, and if that is advantageous in avoiding low-temperature damage. Cultivars that were mildly photoperiod-sensitive were identified from sowing to flowering and from panicle initiation to flowering. The crop models were run for 47 years of temperature data to quantify the risk of encountering low temperature during the critical young microspore stage for 5 different sowing dates. Cultivars that were mildly photoperiod-sensitive, such as Amaroo, had a reduced likelihood of encountering low temperature for a wider range of sowing dates compared with photoperiod-insensitive cultivars. The benefits of increased photoperiod sensitivity include greater sowing flexibility and reduced water use as growth duration is shortened when sowing is delayed. Determining the optimal sowing date also requires other considerations, e. g. the risk of cold damage at other sensitive stages such as flowering and the response of yield to a delay in flowering under non-limiting conditions. It was concluded that appropriate sowing time and the use of photoperiod-sensitive cultivars can be advantageous in the Riverina region in avoiding low temperature damage during reproductive development.

Minimising cold damage during reproductive development among temperate rice genotypes. II. Genotypic variation and flowering traits related to cold tolerance screening

Low temperature during microspore development increases spikelet sterility and reduces grain yield in rice (Oryza sativa L.). The objectives of this study were to determine genotypic variation in spikelet sterility in the field in response to low-temperature and then to examine the use of physio-morphological traits at flowering to screen for cold tolerance. Multiple-sown field experiments were conducted over 4 consecutive years in the rice-growing region of Australia to increase the likelihood of encountering low-temperature during microspore development. More than 50 cultivars of various origins were evaluated, with 7 cultivars common to all 4 years. The average minimum temperature for 9 days during microspore development was used as a covariate in the analysis to compare cultivars at a similar temperature. The low-temperature conditions in Year 4 identified cold-tolerant cultivars such as Hayayuki and HSC55 and susceptible cultivars such as Sasanishiki and Doongara. After low temperature conditions, spikelet sterility was negatively correlated with the number of engorged pollen grains, anther length, anther area, anther width, and stigma area. The number of engorged pollen grains and anther length were found to be facultative traits as their relationships with spikelet sterility were identified only after cold water exposure and did not exist under non-stressed conditions.

The inhibitory effects of free nitrous acid on the energy generation and growth processes of an enriched Nitrobacter culture

The inhibitory effects of nitrite (NO2-)/free nitrous acid (HNO2-FNA) on the metabolism of Nitrobacter were investigated using a method allowing the decoupling of the growth and energy generation processes. A lab-scale sequencing batch reactor was operated for the enrichment of a Nitrobacter culture. Fluorescent in situ hybridization (FISH) analysis showed that 73% of the bacterial population was Nitrobacter. Batch tests were carried out to assess the oxygen and nitrite consumption rates of the enriched culture at low and high nitrite levels, in the presence or absence of inorganic carbon. It was observed that in the absence of CO2, the Nitrobacter culture was able to oxidize nitrite at a rate that is 76% of that in the presence of CO2, with an oxygen consumption rate that is 85% of that measured in the presence of CO2. This enabled the impacts of nitrite/FNA on the catabolic and anabolic processes of Nitrobacter to be assessed separately. FNA rather than nitrite was likely the actual inhibitor to the Nitrobacter metabolism. It was revealed that FNA of up to 0.05 mg HNO2-N center dot L-1 (3.4 mu M), which was the highest FNA concentration used in this study, did not have any inhibitory effect on the catabolic processes of Nitrobacter. However, FNA initiated its inhibition to the anabolic processes of Nitrobacter at approximately 0.011 mg HNO2-N center dot L-1 (0.8 mu M), and completely stopped biomass synthesis at a concentration of approximately 0.023 mg HNO2-N center dot L-1 (1.6 mu M). The inhibitory effect could be described by an empirical inhibitory model proposed in this paper, but the underlying mechanisms remain to be revealed.

Toposequential effects on water balance and productivity in rainfed lowland rice ecosystem in Southern Laos

In the Mekong region, most paddies in rainfed lowland rice (Oryza sativa L.) lie in a sequence on gentle sloping land, and grain yield (GY) often depends on the toposequence position. There is, however, lack of information on toposequential effects on field water supply in rainfed lowland rice and how that influences GY. A total of eight field experiments were carried out on sandy, coarse-textured soils in Southern Laos (Champassak Province and Savannakhet Province) over three wet seasons (2000-2002). Components of the water balance, including downward and lateral water movement (D and L, respectively), were quantified at three different positions along toposequences (top, middle and bottom). GY, days-to-flower (DTF) and rainfall were measured, and the water productivity (WP) was determined. In most experiments, standing water disappeared first in the top position and gradually in lower positions. This was associated with the observation that when there was standing water in the field, the higher position had larger D in both the provinces and also larger L in Champassak Province. However, in one experiment, water loss appeared later in the higher position, as the result of lower L, apparently due to some water inputs other than rainfall occurring at this position. Despite larger D plus L at the top position, seasonal sum of D and L were not much affected by the toposequence position, as the daily rate of D plus L became minimal when the standing water was lost earlier in the top position. Lower GY was associated with earlier disappearance of standing water from the field. Relatively low GY was expected in the top toposequence position. This was clearly shown in the toposequence of Phonthong, Champassak Province, as the timing of standing water disappearance relative to flowering was earlier in the top position. Variation in GY across the toposequence positions was coupled with the WP variation, and both GY and WP tended to decline with increased DTF. Therefore, variation in productivity of rainfed lowland rice across toposequence positions depends mainly on the field water status around flowering time. (c) 2005 Elsevier B.V. All rights reserved.

Use of drought response index for identification of drought tolerant genotypes in rainfed lowland rice

Drought is a major constraint for rice production in the rainfed lowlands in Southeast Asia and Eastern India. The breeding programs for tainted lowland rice in these regions focus on adaptation to a range of drought conditions. However, a method of selection of drought tolerant genotypes has not been established and is considered to be one of the constraints faced by rice breeders. Drought response index (DRI) is based on grain yield adjusted for variation in potential yield and flowering date, and has been used recently, but its consistency among drought environments and hence its usefulness is not certain. In order to establish a selection method and subsequently to identify donor parents for drought resistance breeding, a series of experiments with 15 contrasting genotypes was conducted under well-watered and managed drought conditions at two sites for 5 years in Cambodia. Water level in the field was recorded and used to estimate the relative water level (WLREL) around flowering as an index of the severity of water deficit at the time of flowering for each entry. This was used to determine if DRI or yield reduction was due to drought tolerance or related to the amount of available water at flowering, i.e. drought escape. Grain yield reduction due to drought ranged from 12 to 46%. The drought occurred mainly during the reproductive phase, while four experiments had water stress from the early vegetative stage. There was significant variation for water availability around flowering among the nine experiments and this was associated with variation in mean yield reduction. Genotypic variation in DRI was consistent among most experiments, and genotypic mean DRI ranged from -0.54 to 0.47 (LSD 5% = 0.47). Genotypic variation in DRI was not related to WLREL around flowering in the nine environments. It is concluded that selection for DRI under drought conditions would allow breeders to identify donor lines with high drought tolerance as an important component of breeding better adapted varieties for the rainfed lowlands; two genotypes were identified with high DRI and low yield reduction and were subsequently used in the breeding program in Cambodia. (c) 2006 Elsevier B.V. All rights reserved.

Obtaining highly enriched cultures of candidatus accumulibacter phosphates through alternating carbon sources

Candidatus Accumulibacter Phosphatis is widely considered to be a polyphosphate accumulating organism (PAO) of prime importance in enhanced biological phosphorus removal (EBPR) systems. This organism has yet to be isolated, despite many attempts. Previous studies on the biochemical and physiological aspects of this organism, as well as its response to different EBPR operational conditions, have generally relied on the use of mixed culture enrichments. One frequent problem in obtaining highly enriched cultures of this organism is the proliferation of glycogen accumulating organisms (GAO) that can compete with PAOs for limited carbon sources under similar operational conditions. In this study, Candidatus Accumulibacter Phosphatis has been enriched in a lab-scale bioreactor to a level greater than 90% as quantified by fluorescence in situ hyrbridisation (FISH). This is the highest enrichment of this organism that has been reported thus far, and was obtained by alternating the sole carbon source in the feed between acetate and propionate every one to two sludge ages, and operating the bioreactor within a pH range of 7.0-8.0. Simultaneously, the presence of two known groups of GAOs was eliminated under these operational conditions. Excellent phosphorus removal performance and stability were maintained in this system, where the phosphorous concentration in the effluent was below 0.2 mg/L for more than 7 months. When a disturbance was introduced to this system by adding sludge from an enriched GAO culture, Candidatus Accumulibacter Phosphatis once again became highly enriched, while the GAOs were out-competed. This feeding strategy is recommended for future studies focused on describing the physiology and biochemistry of Accumulibacter, where a highly-enriched culture of this organism is of high importance. (c) 2006 Elsevier Ltd. All rights reserved.