Search

Cbfa2 (AML1) encodes the DNA-binding subunit of a transcription factor in the small family of core-binding factors (CBFs). Cbfa2 is required for the differentiation of all definitive hematopoietic cells, but not for primitive erythropoiesis. Here we show that Cbfa2 is expressed in definitive hematopoietic progenitor cells, and in endothelial cells in sites from which these hematopoietic cells are thought to emerge. Endothelial cells expressing Cbfa2 are in the yolk sac, the vitelline and umbilical arteries, and in the ventral aspect of the dorsal aorta in the aorta/genital ridge/mesonephros (AGM) region. Endothelial cells lining the dorsal aspect of the aorta, and elsewhere in the embryo, do not express Cbfa2. Cbfa2 appears to be required for maintenance of Cbfa2 expression in the endothelium, and for the formation of intra-aortic hematopoietic clusters from the endothelium.

Core-binding factors (CBF) are heteromeric transcription factors essential for several developmental processes, including hematopoiesis. CBFs contain a DNA-binding CBF alpha subunit and a non-DNA binding CBF beta subunit that increases the affinity of CBF alpha for DNA. We have developed a procedure for overexpressing and purifying full-length CBF beta as well as a truncated form containing the N-terminal 141 amino acids using a novel glutaredoxin fusion expression system. Substantial quantities of the CBF beta proteins can be produced in this manner allowing for their biophysical characterization. We show that the full-length and truncated forms of CBF beta bind to a CBF alpha DNA complex with very similar affinities. Sedimentation equilibrium measurements show these proteins to be monomeric. Circular dichroism spectroscopy demonstrates that CBF beta is a mixed alpha/beta protein and NMR spectroscopy shows that the truncated and full-length proteins are structurally similar and suitable for structure determination by NMR spectroscopy.

The Runt domain is the DNA-binding domain defining a small family of transcription factors that are involved in important developmental processes. Developmental pathways controlled by Runt domain proteins include sex determination, neurogenesis, segmentation, and eye development in Drosophila and hematopoiesis in mammals. In addition to binding DNA, the Runt domain also mediates heterodimerization with another subunit called the core-binding factor beta (CBFbeta) subunit. In this study we overexpress the Runt domain from the mouse CBFalpha2 (AML1) protein in Escherichia coli, and purify it from the insoluble fraction. We determine the equilibrium constants for Runt domain binding to two different DNA sequences by surface plasmon resonance technology. Circular dichroism spectroscopy demonstrates that the Runt domain is a folded beta-domain with essentially no alpha-helical content. The single tryptophan residue in the CBFalpha2 Runt domain at amino acid 79 is shown by tryptophan fluorescence spectroscopy to reside in a polar environment. Finally, we demonstrate that ATP can be UV cross-linked to the Runt domain and that ATP binding is sensitive to an amino acid substitution in the putative Kinase-1a motif (P-loop).

Binding sites for six distinct nuclear factors on the 75-base-pair repeat of the Moloney murine leukemia virus enhancer have been identified by an electrophoretic mobility shift assay combined with methylation interference. Three of these factors, found in WEHI 231 nuclear extracts, which we have named LVa, LVb, and LVc (for leukemia virus factors a, b, and c) have not been previously identified. Nuclear factors that bind to the conserved simian virus 40 corelike motif, the NF-1 motif, and the glucocorticoid response element were also detected. Testing of multiple cell lines showed that most factors appeared ubiquitous, except that the NF-1 binding factor was found neither in nuclear extracts from MEL cells nor in the embryonal carcinoma cell lines PCC4 and F9, and core-binding factor was relatively depleted from MEL and F9 nuclear extracts.

Previous studies have demonstrated that the B cells in immune and nonimmune mice manifest different major histocompatibility complex (MHC) collaborative phenotypes with antigen-specific T cells. Immune, or secondary B cells require syngeneic-like MHC recognition by collaborating T cells, and in its absence fail to be stimulated. Primary B cells manifest a much less stringent requisite for MHC recognition by T cells, and under conditions in which secondary B cells fail to be stimulated, primary B cells are stimulated to secrete IgM antibody. Experiments were conducted to determine whether the acquisition of the secondary B cells' MHC collaborative phenotype was dependent on the presence of T cells during in vivo immunization. B cell populations from T dependently and T independently immunized conventional BALB/c and athymic BALB/c nu/nu mice were compared in their ability to collaborate with allogeneic T cells. Although antigen alone promotes the differentiation of several secondary B cell characteristics, including an increase in the frequency of antigen-specific B cells and a preference for IgG1 antibody synthesis in vitro, the acquisition of the secondary B cells' MHC collaborative phenotype was dependent on the presence of T cells during in vivo immunization. B cell populations from T dependently and T independently immunized conventional BALB/c and athymic BALB/c nu/nu mice were compared in their ability to collaborate with allogeneic T cells. Although antigen alone promotes the differentiation of several secondary B cell characteristics, including an increase in the frequency of antigen-specific B cells and a preference of IgG1 antibody synthesis in vitro, the acquisition of the secondary B cells' MHC collaborative phenotype was found to be dependent on the presence of T cells during in vivo immunization. Thus, the restriction imposed on T cell-B-cell-collaborative interactions in secondary humoral immune responses appears to be the result of T dependent antigen-driven events.

Immunization of BALB/c mice with phosphorylcholine-Limulus polyphemus hemocyanin (PC-Hy) induces a population of T cells that recognize the predominant PC-binding antibody, TEPC15 (T15). The splenic fragment culture system was used to examine the specificity of these T cells for a series of PC-binding myeloma and hybridoma antibodies representing the prototype variable region of the heavy chain (VH)T 15 sequence as well as somatic variants of the T15 germ line-encoded sequence. Included in this group of PC-binding proteins were both T15-positive and T15-negative antibodies, as defined by anti-idiotypic antibody. T cell help was identified by the ability to promote TNP-specific B cell responses to trinitrophenylated PC-binding proteins. It was found that T cells generated by immunization with PC-Hy recognize both antibodies with the T15 prototype sequence and the putative somatic variants of this sequence. A population of these T cells appear to recognize common determinants shared by these proteins because immunization with T15 itself also induces the recognition of the somatic variants. This suggests that idiotopes encoded in the T15 germ line gene expressed by the T15 prototype idiotype and the somatic variants can function as targets for T cell recognition and are thus regulatory idiotopes.

IntroductionMalignant pleural effusions are a common complication of advanced malignancy, have a poor prognosis and have a significant impact on quality of life. Treatment strategies include chest drain and pleurodesis, or insertion of an indwelling pleural catheter. Successful pleurodesis is thought to be due to the body’s inflammatory response resulting in pleural symphysis. This post hoc analysis of data from the TIME 1 was conducted to address assess whether there is a correlation between the pleurodesis and a systemic inflammatory response.MethodsA total of 282 patients from the TIME 1 trial had data on pleurodesis success, which was defined as no further pleural procedures for up to 3 months after pleurodesis. Patients who had undergone thoracosopy and poudrage as well as those who had undergone chest drain with pleurodesis were included. Sterile talc was used in all patients. The difference in the white cell count (WCC) and C-reactive protein (CRP) levels was calculated between the day of pleurodesis (Day 0) and Day 1. The data are normally distributed thus independent t test was used for analysis. The CRP Day 0 and 1 data were not normally distributed, and therefore were log transformed to produce a normal distribution.ResultsTwo hundred and eighty two patients were included in the analysis with a mean age of 71 in both groups. 229 had a successful pleurodesis and 53 patients required a further pleural procedure on the ipsilateral side signifying failed pleurodesis. 193 patients had CRP levels and 220 patients had WCC levels recorded on both Day 0 and Day 1.Patients who had a successful pleurodesis had a significantly greater rise in CRP than those who failed pleurodesis. There was no significant difference in the change in WCC between the groups. There was also no significant difference in Day 0 and Day 1 WCC or CRP levels between the two groups.ConclusionsThis analysis demonstrates that systemic rise in CRP as an indicator of inflammation is a better predictor of pleurodesis success than the WCC. These data support the hypothesis that higher levels of inflammation are associated with pleurodesis success.Abstract S129 Table 1Pleurodesis SuccessPleurodesis FailureSignificanceWCC Day 08.84 (SD 4.00, n=213)9.12 (SD – 3.14, n=46)p=0.582WCC Day 111.14 (SD 3.78, n=191)10.71 (SD 4.01, n=42)p=0.525WCC Change2.30 (SD 3.07, n=180)1.55 (SD 2.82, n=40)p=0.140CRP Day 0 (log)1.46 (SD 0.58, n=181)1.45 (SD 0.58, n=42)p=0.900CRP Day 1 (log)1.92 (SD 0.34, n=179)1.83 (SD 0.33, n=41)p=0.123CRP Change47.81 (SD 52.08, n=154)27.05 (SD 32.47, n=39)p=0.003SD=Standard Deviation and n=number of patients