Escherichia coli integration host factor bends the DNA at the ends of IS1 and in an insertion hotspot with multiple IHF binding sites.

The integration host factor of Escherichia coli (IHF) is a small, histone-like protein which participates in the integration of bacteriophage lambda into the E. coli chromosome and in a number of regulatory processes. Our recent footprinting analysis has shown that IHF binds specifically to the ends of the transposable element IS1, as well as to several sites within a short segment of the plasmid pBR322. We have extended our studies of the binding of the IHF molecule to these sites in vitro using a gel retardation assay. We report here that IHF bends the DNA upon binding, as judged from the strong cyclic dependence of the protein-induced mobility shift on the position of the binding site. Using cloned, synthetic ends of IS1 as substrates, we have found that some mutations within the conserved bases of the IHF consensus binding sequence abolish binding, and that alterations of the flanking sequences can greatly reduce IHF binding. The presence of multiple IHF sites on a single DNA fragment increases binding very little, indicating that IHF does not bind cooperatively in this complex. We discuss the possibility that DNA bending is related to the role IHF plays in forming and stabilizing nucleoprotein complexes, and suggest that bending at the IHF sites may be important to its diverse effects in the cell.     

Bovine spermatozoal head size variation and evaluation of a separation technique based on this size

The objectives of this study were to determine the variation of head areas of normal spermatozoa attributable to breed, individual bull and ejaculate and to verify separation of X and Y chromosome-bearing spermatozoa and separation effectiveness. Spermatozoa were evaluated using video enhanced contrast microscopy combined with video intensified fluorescent microscopy and the polymerase chain reaction (PCR). In Experiment 1, spermatozoal head areas were measured from 2 ejaculates collected from bulls of 3 beef and 2 dairy breeds. No differences in head areas were found between breeds or between bulls within breeds; variation was observed among ejaculates from individual bulls across breeds. In Experiment 2, spermatozoa from 5 ejaculates were separated on individual SEPDEVICEs (Patented). Head area, fluorescent intensity and PCR of spermatozoa retained in the SEPDEVICEs suggested a separation based on size in 1 of 5 samples. Ejaculate variation in head areas affected separation efficiency.     

Automated purification and suspension array detection of 16S rRNA from soil and sediment extracts by using tunable surface microparticles

Autonomous, field-deployable molecular detection systems require seamless integration of complex biochemical solutions and physical or mechanical processing steps. In an attempt to simplify the fluidic requirements for integrated biodetection systems, we used tunable surface microparticles both as an rRNA affinity purification resin in a renewable microcolumn sample preparation system and as the sensor surface in a flow cytometer detector. The tunable surface detection limits in both low- and high-salt buffers were 1 ng of total RNA ( approximately 10(4) cell equivalents) in 15-min test tube hybridizations and 10 ng of total RNA ( approximately 10(5) cell equivalents) in hybridizations with the automated system (30-s contact time). RNA fragmentation was essential for achieving tunable surface suspension array specificity. Chaperone probes reduced but did not completely eliminate cross-hybridization, even with probes sharing <50% identity to target sequences. Nonpurified environmental extracts did not irreparably affect our ability to classify color-coded microparticles, but residual environmental constituents significantly quenched the Alexa-532 reporter fluor. Modulating surface charge did not influence the interaction of soluble environmental contaminants with conjugated beads. The automated system greatly reduced the effects of fluorescence quenching, especially in the soil background. The automated system was as efficacious as manual methods for simultaneous sample purification, hybridization, and washing prior to flow cytometry detection. The implications of unexpected target cross-hybridization and fluorescence quenching are discussed relative to the design and implementation of an integrated microbial monitoring system.     

Enterococcal peptide sex pheromones: synthesis and control of biological activity

The enterococcal pheromone-inducible plasmids such as pCF10 represent a unique class of mobile genetic elements whose transfer functions are induced by peptide sex pheromones. These pheromones are excreted by potential recipient cells and detected by plasmid-containing donor cells at the cell surface, where the pheromone is imported and signals induction of the plasmid transfer system. Pheromone is processed from a chromosomally encoded lipoprotein and excreted by both the donor and recipient cells, but a carefully controlled detection system prevents a response to self-pheromone while still allowing an extremely sensitive response to exogenous pheromone.     

Production of huitlacoche, Ustilago maydis: timing inoculation and controlling pollination

Huitlacoche is the name given to young, fleshy, edible galls that form when ears of Zea mays are infected by Ustilago maydis. Huitlacoche is processed and sold fresh at markets in Mexico. Interest has increased recently in producing U. maydis as a specialty mushroom in the United States. Silk-channel inoculation methods developed to evaluate common smut resistance in maize can be used to produce huitlacoche commercially. This research assessed the effects of time of inoculation and preventing pollination on the severity of ear galls and yield of huitlacoche produced by inoculating silks with U. maydis. Yield of huitlacoche and severity of ear galls were closely related, as was evident from highly significant linear or curvilinear regressions. Severity and yield were greatest when ears were inoculated 4-8 d after the mid-silk growth stage. Ear galls were 5-15% more severe and yield of huitlacoche was 18-150% greater on ears that were not pollinated, compared to those that were pollinated. Maximum yield of huitlacoche was 131 g ear(-1) from unpollinated male-sterile field corn inoculated 6 d after the mid-silk growth stage and 92 g ear(-1) from detasseled sweet corn inoculated 6 d after mid-silk. About 25% of the total weight of ears consisted of marketable huitlacoche when yields were highest. Quality of huitlacoche was not affected by time of inoculation or pollination treatments, but quality of huitlacoche harvested 12-14 d after inoculation was unacceptable primarily due to lack of teliospores, which affected color and flavor.     

Cells expressing indoleamine 2,3-dioxygenase inhibit T cell responses

Pharmacological inhibition of indoleamine 2,3-dioxygenase (IDO) activity during murine gestation results in fetal allograft rejection and blocks the ability of murine CD8(+) dendritic cells to suppress delayed-type hypersensitivity responses to tumor-associated peptide Ags. These observations suggest that cells expressing IDO inhibit T cell responses in vivo. To directly evaluate the hypothesis that cells expressing IDO inhibit T cell responses, we prepared IDO-transfected cell lines and transgenic mice overexpressing IDO and assessed allogeneic T cell responses in vitro and in vivo. T cells cocultured with IDO-transfected cells did not proliferate but expressed activation markers. The potency of allogeneic T cell responses was reduced significantly when mice were preimmunized with IDO-transfected cells. In addition, adoptive transfer of alloreactive donor T cells yielded reduced numbers of donor T cells when injected into IDO-transgenic recipient mice. These outcomes suggest that genetically enhanced IDO activity inhibited T cell proliferation in vitro and in vivo. Genetic manipulation of IDO activity may be of therapeutic utility in suppressing undesirable T cell responses.     

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.