Identification of mutants of Arabidopsis defective in acclimation of photosynthesis to the light environment

In common with many other higher plant species, Arabidopsis undergoes photosynthetic acclimation, altering the composition of the photosynthetic apparatus in response to fluctuations in its growth environment. The changes in photosynthetic function that result from acclimation can be detected in a noninvasive manner by monitoring chlorophyll (Chl) fluorescence. This technique has been used to develop a screen that enables the rapid identification of plants defective at ACCLIMATION OF PHOTOSYNTHESIS TO THE ENVIRONMENT (APE) loci. The application of this screen to a population of T-DNA-transformed Arabidopsis has successfully led to the identification of a number of mutant lines with altered Chl fluorescence characteristics. Analysis of photosynthesis and pigment composition in leaves from three such mutants showed that they had altered acclimation responses to the growth light environment, each having a distinct acclimation-defective phenotype, demonstrating that screening for mutants using Chl fluorescence is a viable strategy for the investigation of acclimation. Sequencing of the genomic DNA flanking the T-DNA elements showed that in the ape1 mutant, a gene was disrupted that encodes a protein of unknown function but that appears to be specific to photosynthetic organisms, whereas the ape2 mutant carries an insertion in the region of the TPT gene encoding the chloroplast inner envelope triose phosphate/phosphate translocator.     

Murine EMT-6 carcinoma: high therapeutic efficacy of microbeam radiation therapy

Microbeam radiation therapy is an experimental modality using parallel arrays of thin (<100 micro m) slices of synchrotron-generated X rays (microplanar beams, microbeams). We used EMT-6 murine mammary carcinoma subcutaneously inoculated in the hind legs of mice to compare the therapeutic efficacies of single-fraction, unidirectional (1) "co-planar" microbeams (an array of vertically oriented microplanar beams), (2) "cross-planar" microbeams (two arrays of parallel microbeams propagated in the same direction, one with vertically and the other with horizontally oriented microplanar beams), and (3) seamless (broad) beams from the same synchrotron source. The microbeams were 90 micro m wide and were spaced 300 micro m on center; the median energy in all beams was 100 or 118 keV. Tumor ablation rates were 4/8, 4/8 and 6/7 for a 410-, 520- and 650-Gy in-slice cross-planar microbeam dose, respectively, and 1/8, 3/8, 3/7 and 6/8 for a 23-, 30-, 38- and 45-Gy broad-beam dose, respectively. When the data were pooled from the three highest doses (same average tumor ablations of 50-60%), the incidences of normal-tissue acute toxicity (moist desquamation and epilation) and delayed toxicity (failure of hair regrowth) were significantly lower for cross-planar microbeams than broad beams (P < 0.025). Furthermore, for the highest doses in these two groups, which also had the same tumor ablation rate (>75%), not only were the above toxicities lower for the cross-planar microbeams than for the broad beams (P < 0.02), but severe leg dysfunction was also lower (P < 0.003). These findings suggest that single-fraction microbeams can ablate tumors at high rates with relatively little normal-tissue toxicity.     

Roles for asparagine endopeptidase in class II MHC-restricted antigen processing

The adaptive immune response depends on the creation of suitable peptides from foreign antigens for display on MHC molecules to T lymphocytes. Similarly, MHC-restricted display of peptides derived from self proteins results in the elimination of many potentially autoreactive T cells. Different proteolytic systems are used to generate the peptides that are displayed as T cell epitopes on class I compared with class II MHC molecules. In the case of class II MHC molecules, the proteases that reside within the endosome/lysosome system of antigen-presenting cells are responsible; surprisingly, however, there are relatively few data on which enzymes are involved. Recently we have asked whether proteolysis is required simply in a generic sense, or whether the action of particular enzymes is needed to generate specific class II MHC-associated T cell epitopes. Using the recently identified mammalian asparagine endopeptidase as an example, we review recent evidence that individual enzymes can make clear and non-redundant contributions to MHC-restricted peptide display.     

Functional expression of murine V2R pheromone receptors involves selective association with the M10 and M1 families of MHC class Ib molecules

The vomeronasal organ (VNO) of the mouse has two neuronal compartments expressing distinct families of pheromone receptors, the V1Rs and the V2Rs. We report here that two families of major histocompatibility complex (MHC) class Ib molecules, the M10 and the M1 families, show restricted expression in V2R-expressing neurons. Our data suggest that neurons expressing a given V2R specifically co-express one or a few members of the M10 family. Biochemical and immunocytochemical analysis demonstrates that in VNO sensory dendrites M10s belong to large multi-molecular complexes that include pheromone receptors and beta2-microglobulin (beta2m). In cultured cells, M10s appear to function as escort molecules in transport of V2Rs to the cell surface. Accordingly, beta2m-deficient mice exhibit mislocalization of V2Rs in the VNO and a specific defect in male-male aggressive behavior. The functional characterization of M10 highlights an unexpected role for MHC molecules in pheromone detection by mammalian VNO neurons.     

The effect of nocturnal sampling on semen quality and the efficiency of collection in bovine species

This study evaluated night and day semen collection regimes in Holstein and Brahman bulls (four bulls of each breed) that were collected weekly, each during a morning and a night collection. Ejaculates (n=64) were obtained via artificial vagina over 4 weeks. The first collection of each week alternated between night and day. Two collection teams were employed. Bull behavior parameters included reaction time to first mount, time to ejaculation, a refractory period test, and a thrust intensity test. The numbers of interruptions were counted as a managerial parameter. Pre-freeze semen parameters included total volume, initial motility and concentration. Post-freeze semen parameters measured were: 0- and 3-h post-thaw motility; percent intact acrosomes; and percent sperm abnormalities. Data were analyzed by least squares methods. The bull within breed effect differed (P<0.05) for behavior parameters. The bull within breed effect for total motile sperm harvested was not significant. The bull within breed response was mixed for post-freeze semen viability parameters. Bull within breed was not significant for sperm abnormalities. The night versus day treatment was significant for the managerial parameter (P=0.002). Although a different collection schedule for Bos indicus cattle was not warranted, the efficiency of the collection process was affected by extraneous environmental conditions.     

Modifications to United States Environmental Protection Agency methods 1622 and 1623 for detection of Cryptosporidium oocysts and Giardia cysts in water

Collaborative and in-house laboratory trials were conducted to evaluate Cryptosporidium oocyst and Giardia cyst recoveries from source and finished-water samples by utilizing the Filta-Max system and U.S. Environmental Protection Agency (EPA) methods 1622 and 1623. Collaborative trials with the Filta-Max system were conducted in accordance with manufacturer protocols for sample collection and processing. The mean oocyst recovery from seeded, filtered tap water was 48.4% +/- 11.8%, while the mean cyst recovery was 57.1% +/- 10.9%. Recovery percentages from raw source water samples ranged from 19.5 to 54.5% for oocysts and from 46.7 to 70.0% for cysts. When modifications were made in the elution and concentration steps to streamline the Filta-Max procedure, the mean percentages of recovery from filtered tap water were 40.2% +/- 16.3% for oocysts and 49.4% +/- 12.3% for cysts by the modified procedures, while matrix spike oocyst recovery percentages ranged from 2.1 to 36.5% and cyst recovery percentages ranged from 22.7 to 68.3%. Blinded matrix spike samples were analyzed quarterly as part of voluntary participation in the U.S. EPA protozoan performance evaluation program. A total of 15 blind samples were analyzed by using the Filta-Max system. The mean oocyst recovery percentages was 50.2% +/- 13.8%, while the mean cyst recovery percentages was 41.2% +/- 9.9%. As part of the quality assurance objectives of methods 1622 and 1623, reagent water samples were seeded with a predetermined number of Cryptosporidium oocysts and Giardia cysts. Mean recovery percentages of 45.4% +/- 11.1% and 61.3% +/- 3.8% were obtained for Cryptosporidium oocysts and Giardia cysts, respectively. These studies demonstrated that the Filta-Max system meets the acceptance criteria described in U.S. EPA methods 1622 and 1623.     

Comparison of ribotyping and repetitive extragenic palindromic-PCR for identification of fecal Escherichia coli from humans and animals

This report compares the performances of two popular genotypic methods used for tracking the sources of fecal pollution in water, ribotyping and repetitive extragenic palindromic-PCR (rep-PCR). The rep-PCR was more accurate, reproducible, and efficient in associating DNA fingerprints of fecal Escherichia coli with human and animal hosts of origin.     

Analysis of body segment parameter differences between four human populations and the estimation errors of four popular mathematical models

Calculating the kinetics of motion using inverse or forward dynamics methods requires the use of accurate body segment inertial parameters. The methods available for calculating these body segment parameters (BSPs) have several limitations and a main concern is the applicability of predictive equations to several different populations. This study examined the differences in BSPs between 4 human populations using dual energy x-ray absorptiometry (DEXA), developed linear regression equations to predict mass, center of mass location (CM) and radius of gyration (K) in the frontal plane on 5 body segments and examined the errors produced by using several BSP sources in the literature. Significant population differences were seen in all segments for all populations and all BSPs except hand mass, indicating that population specific BSP predictors are needed. The linear regression equations developed performed best overall when compared to the other sources, yet no one set of predictors performed best for all segments, populations or BSPs. Large errors were seen with all models which were attributed to large individual differences within groups. Equations which account for these differences, including measurements of limb circumferences and breadths may provide better estimations. Geometric models use these parameters, however the models examined in this study did not perform well, possibly due to the assumption of constant density or the use of an overly simple shape. Creating solids which account for density changes or which mimic the mass distribution characteristics of the segment may solve this problem. Otherwise, regression equations specific for populations according to age, gender, race, and morphology may be required to provide accurate estimations of BSPs for use in kinetic equations of motion.