Objective The transcription factor PU.1 (encoded by Sfpi1) promotes myeloid differentiation but it is unclear what downstream genes are involved. MiRNAs are a class of small RNAs that regulate many cellular pathways including proliferation, survival and differentiation. The objective of this study was to identify miRNAs downstream of PU.1 that regulate hematopoietic development. Materials and Methods MiRNAs that change expression in a PU.1-inducible cell line were identified with microarrays. The promoter for a miRNA cluster upregulated by PU.1 induction was analyzed for PU.1 binding by electrophoretic mobility shift and chromatin immunoprecipitation assays. Retroviral transduction of hematopoietic progenitors was performed to evaluate the effect of miRNA expression on hematopoietic development in vitro and in vivo. Results We identified a miRNA cluster whose pri-transcript is regulated by PU.1. The pri-miRNA encodes three mature miRNAs: miR-23a, miR-27a, and miR-24-2. Each miRNA is more abundant in myeloid cells compared to lymphoid cells. When hematopoietic progenitors expressing the 23a cluster miRNAs were cultured in B cell promoting conditions we observed a dramatic decrease in B lymphopoiesis and an increase in myelopoiesis compared to control cultures. In vivo, hematopoietic progenitors expressing the miR-23a cluster generate reduced numbers of B cells compared to control cells. Conclusions The miR-23a cluster is a downstream target of PU.1 involved in antagonizing lymphoid cell fate acquisition. Although miRNAs have been identified downstream of PU.1 in mediating the development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in regulating the development of myeloid versus lymphoid cells.

Neurobehavioral deficits in higher cortical systems have not been described previously in a large animal model of diffuse brain injury. Anesthetized 3-5 day old piglets were subjected to either mild (142 rad/sec) or moderate (188 rad/sec) rapid non-impact axial rotations of the head. Multiple domains of cortical function were evaluated 5 times during the 12 day post-injury period using tests of neurobehavioral function devised for piglets. There were no observed differences in neurobehavioral outcomes between mild injury pigs (N = 8) and instrumented shams (N = 4). Moderately injured piglets (N = 7) had significantly lower interest in exploring their environment and had higher failure rates in visual-based problem solving compared to instrumented shams (N = 5) on Day 1 and 4 after injury. Neurobehavioral functional deficits correlated with neuropathologic damage in the neonatal pigs after inertial head injury. Injured axons detected by immunohistochemistry (β-APP) were absent in mild injury and sham piglets, but were observed in moderately injured piglet brains. In summary, we have developed a quantitative battery of neurobehavioral functional assessments for large animals that correlate with neuropathologic axonal damage and may have wide applications in the fields of cardiac resuscitation, stroke, and hypoxicischemic brain injury.

Mice lacking the zinc finger transcriptional repressor protein GFI-1 are neutropenic. These mice generate abnormal immature myeloid cells exhibiting characteristics of both macrophages and granulocytes. Furthermore, Gfi-1 ؊/؊ mice are highly susceptible to bacterial infection. Interestingly, Gfi-1 ؊/؊ myeloid cells overexpress target genes of the PU.1 transcription factor such as the macrophage colony-stimulating factor receptor and PU.1 itself. We therefore determined whether GFI-1 modulates the transcriptional activity of PU.1. Our data demonstrate that GFI-1 physically interacts with PU

The transcription factor Foxp3 is critical to the suppressive phenotype of CD4+ regulatory T cells. Studies have clearly shown that numerous autoimmune diseases are marked by the presence of activated CD4+ T cells within the setting of chronic inflammation. Therefore drugs capable of inducing Foxp3 expression in activated CD4+ T cells could be of great therapeutic interest. We have previously shown that the small molecule G-1, an agonist directed against the membrane-bound estrogen receptor GPER, can induce IL10 expression in naïve CD4+ T cells. In addition, we and others have demonstrated that G-1 attenuates disease in an animal model of experimental autoimmune encephalomyelitis. Using ex vivo cultures of purified CD4+ T cells, we show that G-1 can elicit Foxp3 expression under TH17 polarizing conditions, which mimic the in situ inflammatory milieu of several autoimmune diseases. These findings build upon previous results demonstrating the immunosuppressive properties of the novel estrogenic small molecule G-1.

The phosphoenolpyruvate:sugar phospho- MATERIALS AND METHODS Growth of E. coil Strains. The strains of E. coli used in this study are listed in Table 1. The plasmid pDIA100 (10) encodes the gene for adenylate cyclase; the plasmids pEL06 (9) and pDS20 (11) encode genes for the PTS proteins HPr, enzyme I, and a fragment of enzyme iiiGIc; and plasmid pDS48 (11) encodes the gene for enzyme IIIGIc. The plasmid pEL06 resident in strain 600 results in an approximately 20-to 70-fold overexpression of enzyme I (9, 11). Strains were grown in LB medium (12) at 370C with aeration. For studies involving the localization of 83-galactosidase, cells were grown to midlogarithmic phase, at which point 5 mM isopropyl B-D-thiogalactopyranoside was added and the cultures were allowed to grow for another hour. Plasmids were maintained in the appropriate strains by including in the cultures ampicillin (30 ,Ag/ml), kanamycin (50 jug/ml), or tetracycline (10 kg/ml). Overnight cultures of the strains were diluted 1:10 with fresh medium and grown for 4 hr. The cells were collected by centrifugation (10,000 x g) and then washed with phosphate-buffered saline (PBS) at pH 7.2. The washed cells were resuspended in 0.5% glutaraldehyde/0.6% tannic acid in PBS for 30 min at room temperature (7,8). The fixed cells were washed once with PBS followed by several washes in 30% (vol/vol) glycerol in PBS or 2.3 M sucrose in PBS for cryoprotection (13). After the final wash, the wet pellet was thoroughly mixed with a Vortex mixer. Drops of the thick suspension were placed on cryomicrotome specimen pins and quickly frozen by plunging them into liquid nitrogen-cooled propane (14, 15). These specimens were stored in a liquid nitrogen reservoir for future sectioning.Enzyme I and Antibody Reagents. Enzyme I was purified from E. coli strain 599 (9) as described (16). Samples of the purified protein were used for production of rabbit polyclonal antibody by standard procedures.fi-Galactosidase. The f3-galactosidase activity of strains KL16 and 600 were measured by the method of Miller (17). The activities were strain KL16 = 185 Miller units and strain 600 = 69 Miller units. Rabbit antibody against E. coli ,3-galactosidase was from Cappell Laboratories.Immunoelectron Microscopy. Specimen pins were mounted on the holder of a Cryonova cryomicrotome (LKB) and 60-to 80-nm-thick sections were cut with a freshly broken glass knife at -98°C. Ribbons of dry sections were collected from the edge of the knife on freezing drops of sucrose (13); they were then transferred to Formvar-coated electron microscope grids, which were placed on liquefied gelatin. Immunolabeling of the sections for revealing antigenic sites and staining of the sections on the grids for generating contrast of the cellular structures were accomplished by floating the grids on drops of solutions placed in Petri dishes. The treatments were done in the following sequence (all solutions were made in PBS): 0.02 M glycine, preimmune serum (1: 1000 dilution), immune serum (1:1000 dilution), protein A...

Antagonistic interactions between transcription factors contribute to cell fate decisions made by multipotent hematopoietic progenitor cells. Concentration of the transcription factor PU.1 affects myeloid/lymphoid development with high levels of PU.1 directing myeloid cell fate acquisition at the expense of B cell differentiation. High levels of PU.1 may be required for myelopoiesis in order to overcome inhibition of its activity by transcription factors that promote B cell development. The B cell transcription factors, E2A and EBF, are necessary for commitment of multipotential progenitors and lymphoid primed multipotential progenitors to lymphocytes. In this report we hypothesized that factors required for early B cell commitment would bind to PU.1 and antagonize its ability to induce myeloid differentiation. We investigated whether E2A and/or EBF associate with PU.1. We observed that the E2A component, E47, but not EBF, directly binds to PU.1. Additionally E47 represses PU.1-dependent transactivation of the MCSFR promoter through antagonizing PU.1's ability to bind to DNA. Exogenous E47 expression in hematopoietic cells inhibits myeloid differentiation. Our data suggest that E2A antagonism of PU.1 activity contributes to its ability to commit multipotential hematopoietic progenitors to the lymphoid lineages.

A protein with a tetragonal pattern, defined as RS protein, was found on the wall surface of an alkaline phosphatase secretion-deficient mutant (NM 105) of Bacillus licheniformis 749/C. The protein was present on the wall surface of the exponential-growth-phase cells, but at the stationary growth phase it was overproduced and hypersecreted. This protein was precipitated to homogeneity from the culture fluid by 80% ammonium sulfate saturation and chilled acetone. The molecular mass of the protein was 98 kilodaltons, and it had a single subunit in a sodium dodecyl sulfate gel. Specific anti-RS antibody was generated in rabbits and used to immunolabel the RS protein on the cells at different growth phases. In early-exponential-growth-phase cells, the outside surface of the wail, the cytoplasm, and the inside surface of the cytoplasmic membrane were labeled.In stationary-growth-phase cells, the cytoplasm was poorly labeled, but the labeling on the outside surface of the wall was high. A B. licheniformis NM 105 gene library was made by using the lambda phage EMBL3. The RS protein expression from this gene library was detected by a modified autoradiographic procedure. One of the amplified RS protein-positive plaques (4213-1) containing recombinant DNA was chosen, and the restriction map of this DNA was prepared. The RS protein expressed in Escherichia coli NM 539 infected with 4213-1 recombinant phage had a lower molecular mass than the purified authentic RS protein. The 4.5-kilobase-pair (kbp) Sall-EcoRI fragment of the recombinant DNA was cloned in the shuttle plasmid pMK4 to construct pMK462, which was expressed in B. subtilis MI112 and produced the RS protein identical in molecular mass to the purified authentic RS protein. The

Background Glass slide preparations from a variety of specimens (blood, masses, effusions) are commonly made as part of the diagnostic work-up, however the effects of various drying methods in veterinary practice and diagnostic laboratory settings is not clear. Objective Compare the effects of four drying methods on results of microscopic examination of canine blood smears and direct smears of pleural or peritoneal effusion fluid. Methods Twelve canine blood samples (6 from healthy dogs, 6 from sick dogs) and 6 canine peritoneal or pleural effusion samples. Four smears were prepared from each of the 18 samples and dried using the following methods: air-dry, hair dryer with or without heat, and heat block at 58 °C. Observers, blinded to the drying method, independently reviewed the slides microscopically, using a scoring system to evaluate cell morphology and (for blood smears) echinocyte numbers; scoring results were analyzed statistically. Results For blood smears, several comparisons showed more adverse effects on morphology using the heat block method than for one or more other drying methods. For effusion fluid smears, RBCs dried with the heat block or air-dry methods had more poorly preserved morphology than RBCs dried by the hair dryer method without heat. Conclusions and clinical relevance The results (1) indicate that different drying methods had a significant effect, (2) support using a hair dryer without heat for both blood smears and effusion fluid smears, and (3) discourage using a 58 °C heat block.