Robust detection of individual forensic profiles in DNA mixtures.

For a forensic identification method to be admissible in international courts, the probability of false match must be quantified. For comparison of individuals against complex mixtures using a panel of single nucleotide polymorphisms (SNPs), the probability of a random man not excluded, P(RMNE) is one admissible standard. While the P(RMNE) of SNP alleles has been previously studied, it remains to be rigorously defined and calculated for experimentally genotyped mixtures. In this report, exact P(RMNE) values were calculated for a range of complex mixtures, verified with Monte Carlo simulations, and compared alongside experimentally determined detection probabilities.

Multiple functional domains of AML1: PU.1 and C/EBPalpha synergize with different regions of AML1.

Control elements of many genes are regulated by multiple activators working in concert to confer the maximal level of expression, but the mechanism of such synergy is not completely understood. The promoter of the human macrophage colony-stimulating factor (M-CSF) receptor presents an excellent model with which we can study synergistic, tissue-specific activation for two reasons. First, myeloid-specific expression of the M-CSF receptor is regulated transcriptionally by three factors which are crucial for normal hematopoiesis: PU.1, AML1, and C/EBPalpha. Second, these proteins interact in such a way as to demonstrate at least two examples of synergistic activation. We have shown that AML1 and C/EBPalpha activate the M-CSF receptor promoter in a synergistic manner. As we report here, AML1 also synergizes, and interacts physically, with PU. 1. Detailed analysis of the physical and functional interaction of AML1 with PU.1 and C/EBPalpha has revealed that the proteins contact one another through their DNA-binding domains and that AML1 exhibits cooperative DNA binding with C/EBPalpha but not with PU.1. This difference in DNA-binding abilities may explain, in part, the differences observed in synergistic activation. Furthermore, the activation domains of all three factors are required for synergistic activation, and the region of AML1 required for synergy with PU.1 is distinct from that required for synergy with C/EBPalpha. These observations present the possibility that synergistic activation is mediated by secondary proteins contacted through the activation domains of AML1, C/EBPalpha, and PU.1.

PU.1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colony-stimulating factor receptor alpha gene.

Growth factor receptors play an important role in hematopoiesis. In order to further understand the mechanisms directing the expression of these key regulators of hematopoiesis, we initiated a study investigating the transcription factors activating the expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha gene. Here, we demonstrate that the human GM-CSF receptor alpha promoter directs reporter gene activity in a tissue-specific fashion in myelomonocytic cells, which correlates with its expression pattern as analyzed by reverse transcription PCR. The GM-CSF receptor alpha promoter contains an important functional site between positions -53 and -41 as identified by deletion analysis of reporter constructs. We show that the myeloid and B cell transcription factor PU.1 binds specifically to this site. Furthermore, we demonstrate that a CCAAT site located upstream of the PU.1 site between positions -70 and -54 is involved in positive-negative regulation of the GM-CSF receptor alpha promoter activity. C/EBP alpha is the major CCAAT/enhancer-binding protein (C/EBP) form binding to this site in nuclear extracts of U937 cells. Point mutations of either the PU.1 site or the C/EBP site that abolish the binding of the respective factors result in a significant decrease of GM-CSF receptor alpha promoter activity in myelomonocytic cells only. Furthermore, we demonstrate that in myeloid and B cell extracts, PU.1 forms a novel, specific, more slowly migrating complex (PU-SF) when binding the GM-CSF receptor alpha promoter PU.1 site. This is the first demonstration of a specific interaction with PU.1 on a myeloid PU.1 binding site. The novel complex is distinct from that described previously as binding to B cell enhancer sites and can be formed by addition of PU.1 to extracts from certain nonmyeloid cell types which do not express PU.1, including T cells and epithelial cells, but not from erythroid cells. Furthermore, we demonstrate that the PU-SF complex binds to PU.1 sites found on a number of myeloid promoters, and its formation requires an intact PU.1 site adjacent to a single-stranded region. Expression of PU.1 in nonmyeloid cells can activate the GM-CSF receptor alpha promoter. Deletion of the amino-terminal region of PU.1 results in a failure to form the PU-SF complex and in a concomitant loss of transactivation, suggesting that formation of the PU-SF complex is of functional importance for the activity of the GM-CSF receptor alpha promoter. Finally, we demonstrate that C/EBP alpha can also active the GM-CSF receptor alpha promoter in nonmyeloid cells. These results suggest that PU.1 and C/EBP alpha direct the cell-type-specific expression of GM-CSF receptor alpha, further establish the role of PU.1 as a key regulator of hematopoiesis, and point to C/EBP alpha as an additional important factor in this process.

Monocyte chemoattractant protein-1 gene is expressed in activated neutrophils and retinoic acid-induced human myeloid cell lines.

We have used differential display polymerase chain reaction to identify genes that are upregulated after retinoic acid (RA) treatment of human myeloblastic HL-60 cells. Three of the cDNAs cloned hybridized to RA-inducible transcripts on Northern blots, one of which was shown to encode sequences for monocyte chemoattractant protein-1 (MCP-1), a recently described cytokine that is chemotactic for monocytes but not for neutrophils. Nuclear run-on analysis indicated that the upregulation of the MCP-1 gene occurs at the transcriptional level in HL-60 cells. MCP-1 transcript levels also increased after RA treatment of the NB4 acute promyelocytic cell line. MCP-1 transcripts were undetectable in freshly isolated neutrophils by Northern analysis or reverse transcription-polymerase chain reaction but were readily detectable in neutrophils after incubation in media at 37 degrees C for 20 hours, suggesting that an activation event can lead to MCP-1 expression in neutrophils. Immunocytochemistry confirmed the presence of MCP-1 protein in activated neutrophils. This is the first report that the MCP-1 gene is RA-responsive in myeloid cell lines and is expressed in neutrophils. MCP-1 expression by activated neutrophils may play an important role in attracting monocytes to the site of tissue damage or infection.

Carboxyhemoglobin and brain blood flow in humans.

It has been shown that with increased carboxyhemoglobin (COHb) and associated decrease in blood oxygen-carrying capacity, a compensatory increase in brain-blood flow (BBF) develops. The BBF response in humans has been shown to be quite variable. Two experiments were conducted in which humans were exposed to sufficient carbon monoxide (CO) to produce COHb levels up to 18.4%. BBF was measured by the method of impedance plethysmography. The first was a pilot study in which BBF in 14 men was studied after transient exposure to various concentrations of CO in air. BBF increased as a function of COHb but not to the same extent (or at all) in some subjects. In a confirmatory experiment with 12 men, BBF was measured once per h during a 4-h experiment. All 12 subjects received CO. The variation of the BBF response among subjects was large and statistically significant whereas the variation over time was not significant. Thus it appears that the magnitude of the BBF response is unique for a given subject and differs across subjects. These results may help predict CO-induced behavioral decrements in future studies if subjects whose BBF response to COHb is small or absent are also more susceptible to impairment by acute CO exposure.

Continuous noninvasive monitoring of left ventricular function during exercise by thoracic impedance cardiography-automated derivation of systolic time intervals.

Systolic time intervals (STI) obtained during exercise are useful as a method of estimating global left ventricular function. The conventional method, however, which requires a carotid pulse tracing as well as a phonocardiogram of high quality, is technically difficult under conditions of exercise. We have validated a new method of obtaining STI which employs the first derivative of thoracic electrical impedance (DZ/DT). The DZ/DT was recorded using surface electrodes and a microcomputer for automated signal processing. The new method was studied in 20 male normal subjects (aged 18 to 53 years) at rest and during increasing levels of upright exercise. Heart rate ranged from 61 to 173 beats/min. Results obtained simultaneously by both techniques showed no significant difference. Thus impedance cardiography allows continuous monitoring of STI during exercise and may prove to be a valuable addition to multistage stress testing.

A comprehensive cardiac exercise stress processor for environmental health effects studies.

We have shown that an interactive microcomputer system using noninvasive cardiovascular measurements during exercise is both possible and practical. Experimental use of the system has verified our choice of variables as appropriate for automatic generation of a cardiovascular data base, but additional studies are required to determine the system's sensitivity for assessing health-effect decrements.

An analogue echogram range gate tracker for clinical echocardiography.

The fundamental purpose of this paper is to present the results of a feasibility study for an on-line, real-time automatic range gate tracker (ARGT) for clinical echocardiography, specifically for the purpose of determining cardiac output noninvasively and in real time. The basic ARGT design represents an implementation of specific algorithms and a system controller that permit user interaction and interpretation of clinical echocardiograms. In its analytical mode, the ARGT automatically tracks cardiac echoes as visually perceived and selected by the user. While tracking specific cardiac echoes, the ARGT converts the complex ultrasonic echogram into a pair of meaningful analogue signals (corresponding to the motion of the anterior and posterior left ventricular walls) from which dimensional information corresponding to dynamic geometric changes of the heart are derived. From the information, the cardiac output is computed in real time.