Quantification of substrate and cellular strains in stretchable 3D cell cultures: an experimental and computational framework.

The mechanical cell environment is a key regulator of biological processes . In living tissues, cells are embedded into the 3D extracellular matrix and permanently exposed to mechanical forces. Quantification of the cellular strain state in a 3D matrix is therefore the first step towards understanding how physical cues determine single cell and multicellular behaviour. The majority of cell assays are, however, based on 2D cell cultures that lack many essential features of the in vivo cellular environment. Furthermore, nondestructive measurement of substrate and cellular mechanics requires appropriate computational tools for microscopic image analysis and interpretation. Here, we present an experimental and computational framework for generation and quantification of the cellular strain state in 3D cell cultures using a combination of 3D substrate stretcher, multichannel microscopic imaging and computational image analysis. The 3D substrate stretcher enables deformation of living cells embedded in bead-labelled 3D collagen hydrogels. Local substrate and cell deformations are determined by tracking displacement of fluorescent beads with subsequent finite element interpolation of cell strains over a tetrahedral tessellation. In this feasibility study, we debate diverse aspects of deformable 3D culture construction, quantification and evaluation, and present an example of its application for quantitative analysis of a cellular model system based on primary mouse hepatocytes undergoing transforming growth factor (TGF-ß) induced epithelial-to-mesenchymal transition.

Determination of the burden of hepatitis C virus infection in Ireland.

Information on the burden of hepatitis C virus (HCV) disease is needed to inform policy decisions on primary and secondary prevention. Specimen-based laboratory data (1989-2004) were converted to person-based data and combined with notification data (2004-2009) to describe the burden of HCV infection in Ireland. More than 10,000 people were confirmed as HCV infected in 1989-2004, with the numbers peaking in 2000. The predominant genotypes were 1 (55%) and 3 (39%). Drug use was the most likely risk factor in 80%, with receipt of blood or blood products in 16%. It is estimated that 20 000-50,000 people in Ireland are chronically infected with HCV, a population prevalence of 0·5-1·2%, which is similar to other countries in Northern Europe. This is the first published estimate of the number of chronic HCV infections in Ireland. These data will be of value in health service planning and will contribute to the understanding of HCV infection in Europe.

[Personalised hepatology - current concepts, developments and expectations in the post-genome era]. / Personalisierte Hepatologie - Gegenwärtige Konzepte, Entwicklungen und Erwartungen im Postgenomzeitalter.

Promoted by the decoding of the human genome as part of the human genome project, individualised therapy approaches have become a realistic perspective for therapies that are more effective, less prone to side effects and economically reasonable. This also applies to chronic liver disease. With the aim not only to expand the current knowledge base through basic research on the underlying disease processes and treatment options but also to identify and characterise biomarkers, the creation of genetic fingerprints for individualised diagnosis, prognosis and treatment of patients takes its place in the centre of translational hepatology. For certain liver diseases personalised therapy approaches are already existent. Examples are the determination of viral genotypes, viral kinetics and genotyping of the IL28B polymorphism to optimise the treatment of chronic hepatitis C. The challenges of the next few years relate to the broadening of the knowledge base, the establishment of reliable and standardised technologies, and the development of intelligent bioinformatics strategies for data analysis and data integration. The following review not only summarises the current state of progress and possibilities of personalised medicine in hepatological diseases, but also explains the technical background of the limitations that currently hinder a consistent clinical implementation.

Interfering with interferon-γ signalling in intestinal epithelial cells: selective inhibition of apoptosis-maintained secretion of anti-inflammatory interleukin-18 binding protein.

The intestinal epithelial barrier represents an important component in the pathogenesis of inflammatory bowel diseases. Interferon (IFN)-γ, a T helper type 1 (Th1) cytokine, regulated by the interleukin (IL)-18/IL-18 binding protein (bp) system, modulates the integrity of this barrier. The aim of this work was to study functionally the consequences of IFN-γ on intestinal epithelial cells (IEC) and to interfere selectively with identified adverse IFN-γ effects. IEC lines were stimulated with IFN-γ. IL-18 and IL-18bp were assessed by enzyme-linked immunosorbent assay. Staining of phosphatidylserine, DNA laddering, lactate dehydrogenase (LDH) release, cleavage of poly-adenosine diphosphate-ribose-polymerase (PARP) and activation of caspase-3 were analysed to determine cell death. Inhibitors of tyrosine kinase, caspase-3 or p38 mitogen-activated kinase ((MAP) activity were used. Cytokines were measured in supernatants of colonic biopsies of healthy controls and inflammatory bowel disease (IBD) patients. In IEC lines, IFN-γ up-regulated IL-18bp selectively. Ex vivo, IFN-γ was present in supernatants from cultured biopsies and up-regulated with inflammation. Contrary to previous reports, IFN-γ alone induced apoptosis in IEC lines, as demonstrated by phosphatidylserin staining, DNA cleavage and LDH release. Further, activation of caspase-3, PARP cleavage and expression of pro-apoptotic Bad were induced. Partial inhibition of caspase-3 and of p38 but not JAK tyrosine kinase, preserved up-regulation of IL-18bp expression. Selective inhibition of IFN-γ mediated apoptosis, while preserving its beneficial consequences on the ratio of IL-18/IL-18bp, could contribute to the integrity of the mucosal barrier in intestinal inflammation.

An observational study of a cross platform risk assessment mobile application in a forensic inpatient setting.

Consumer-focused healthcare mobile applications have seen widespread adoption in recent years. Enterprise mobile applications in hospital settings have been slower to gain traction. In this study we examine the Dynamic Appraisal of Situational Aggression: Inpatient version (DASA), a short-term risk assessment tool which is well validated and widely used in the prediction of violent incidents, within an inpatient forensic setting. The application was piloted over a period of three months, collecting 847 total DASA scores on 21 different patients. Time stamping allowed for accurate correlation between risk assessment scoring and the violent risk incidents. The internal validity of the app was measured using Cronbach's alpha and was calculated at 0.798 indicating good internal validity. Using violent incidents as the dependent factor and the total DASA score as the independent factor, predictive validity of the app was calculated at 0.85, p = 0.007. The use of this application in a forensic setting was successful with good internal and predictive validity. A major benefit of this form of data collection was the electronic time stamping so that the correlation between risk estimation and events could be more closely correlated. Deployment of such an application in a general hospital setting would bring its own challenges but would be useful in other types of risk assessment and screening tools.

Blood monocyte-derived neohepatocytes as in vitro test system for drug metabolism.

The gold standard for human drug metabolism studies is primary hepatocytes. However, availability is limited by donor organ scarcity. Therefore, efforts have been made to provide alternatives, e.g., the hepatocyte-like (NeoHep) cell type, which was generated from peripheral blood monocytes. In this study, expression and activity of phase I and phase II drug-metabolizing enzymes were investigated during transdifferentiation of NeoHep cells and compared with primary human hepatocytes. Important drug-metabolizing enzymes are cytochrome P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, and 3A4), microsomal epoxide hydrolase 1, glutathione S-transferase A1 and M1, N-acetyltransferase 1, NAD(P)H menadione oxidoreductase 1, sulfotransferase 1A1, and UDP-glucuronosyltransferase 1A6. Monocytes and programmable cells of monocytic origin expressed only a few of the enzymes investigated. Throughout differentiation, NeoHep cells showed a continuously increasing expression of all drug-metabolizing enzymes investigated, resulting in stable basal activity after approximately 15 days. Fluorescence-based activity assays indicated that NeoHep cells and primary hepatocytes have similar enzyme kinetics, although the basal activities were significantly lower in NeoHep cells. Stimulation with 3-methylcholanthrene and rifampicin markedly increased CYP1A1/2 or CYP3A4 activities, which could be selectively inhibited by nifedipine, verapamil, ketoconazole, and quercetin. Our data reveal similarities in expression, activity, induction, and inhibition of drug-metabolizing enzymes between NeoHep cells and primary human hepatocytes and hence suggest that NeoHep cells are useful as an alternative to human hepatocytes for measuring bioactivation of substances.

Effect of Intravenous Administration of Cobalt Chloride to Horses on Clinical and Hemodynamic Variables.

BACKGROUND: Cobalt chloride (CoCl2 ) is administered to racehorses to enhance performance. The purpose of this study was to evaluate the clinical, cardiovascular, and endocrine effects of parenterally administered CoCl2 . OBJECTIVES: To describe the effects of weekly intravenous doses of CoCl2 on Standardbred horses. ANIMALS: Five, healthy Standardbred mares. METHODS: Prospective, randomized, experimental dose-escalation pilot. Five Standardbred mares were assigned to receive 1 of 5 doses of CoCl2 (4, 2, 1, 0.5, or 0.25 mg/kg) weekly IV for 5 weeks. Physical examination, blood pressure, cardiac output, and electrocardiography (ECG) were evaluated for 4 hours after administration of the first and fifth doses. Blood and urine samples were collected for evaluation of cobalt concentration, CBC and clinical chemistry, and hormone concentrations. RESULTS: All mares displayed pawing, nostril flaring, muscle tremors, and straining after CoCl2 infusion. Mares receiving 4, 2, or 1 mg/kg doses developed tachycardia after dosing (HR 60-126 bpm). Ventricular tachycardia was noted for 10 minutes after administration of the 4 mg/kg dose. Increases in systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and mean arterial pressure (MAP) occurred after administration of all doses (4, 2, 1, 0.5, and 0.25 mg/kg). Profound hypertension was observed after the 4 mg/kg dose (SAP/DAP, MAP [mmHg] = 291-300/163-213, 218-279). Hemodynamics normalized by 1-2 hours after administration. ACTH and cortisol concentrations increased within 30 minutes of administration of all CoCl2 doses, and cardiac troponin I concentration increased after administration of the 4 and 2 mg/kg doses. CONCLUSIONS AND CLINICAL IMPORTANCE: The degree of hypertension and arrhythmia observed after IV CoCl2 administration raises animal welfare and human safety concerns.

Pharmacokinetics and pharmacodynamics of buprenorphine and sustained-release buprenorphine after administration to adult alpacas.

OBJECTIVE To determine pharmacokinetics and pharmacodynamics of buprenorphine after IV and SC administration and of sustained-release (SR) buprenorphine after SC administration to adult alpacas. ANIMALS 6 alpacas. PROCEDURES Buprenorphine (0.02 mg/kg, IV and SC) and SR buprenorphine (0.12 mg/kg, SC) were administered to each alpaca, with a 14-day washout period between administrations. Twenty-one venous blood samples were collected over 96 hours and used to determine plasma concentrations of buprenorphine. Pharmacokinetic parameters were calculated by use of noncompartmental analysis. Pharmacodynamic parameters were assessed via sedation, heart and respiratory rates, and thermal and mechanical antinociception indices. RESULTS Mean ± SD maximum concentration after IV and SC administration of buprenorphine were 11.60 ± 4.50 ng/mL and 1.95 ± 0.80 ng/mL, respectively. Mean clearance was 3.00 ± 0.33 L/h/kg, and steady-state volume of distribution after IV administration was 3.8 ± l.0 L/kg. Terminal elimination half-life was 1.0 ± 0.2 hours and 2.7 ± 2.8 hours after IV and SC administration, respectively. Mean residence time was 1.3 ± 0.3 hours and 3.6 ± 3.7 hours after IV and SC administration, respectively. Bioavailability was 64 ± 28%. Plasma concentrations after SC administration of SR buprenorphine were below the LLOQ in samples from 4 alpacas. There were no significant changes in pharmacodynamic parameters after buprenorphine administration. Alpacas exhibited mild behavioral changes after all treatments. CONCLUSIONS AND CLINICAL RELEVANCE Buprenorphine administration to healthy alpacas resulted in moderate bioavailability, rapid clearance, and a short half-life. Plasma concentrations were detectable in only 2 alpacas after SC administration of SR buprenorphine.

Hepatocyte-specific Smad7 deletion accelerates DEN-induced HCC via activation of STAT3 signaling in mice.

TGF-ß signaling in liver cells has variant roles in the dynamics of liver diseases, including hepatocellular carcinoma (HCC). We previously found a correlation of high levels of the important endogenous negative TGF-ß signaling regulator SMAD7 with better clinical outcome in HCC patients. However, the underlying tumor-suppressive molecular mechanisms are still unclear. Here, we show that conditional (TTR-Cre) hepatocyte-specific SMAD7 knockout (KO) mice develop more tumors than wild-type and corresponding SMAD7 transgenic mice 9 months after diethylnitrosamine (DEN) challenge, verifying SMAD7 as a tumor suppressor in HCC. In line with our findings in patients, Smad7 levels in both tumor tissue as well as surrounding tissue show a significant inverse correlation with tumor numbers. SMAD7 KO mice presented with increased pSMAD2/3 levels and decreased apoptosis in the tumor tissue. Higher tumor incidence was accompanied by reduced P21 and upregulated c-MYC expression in the tumors. Activation of signal transducer and activator of transcription factor 3 signaling was found in Smad7-deficient mouse tumors and in patients with low tumoral SMAD7 expression as compared with surrounding tissue. Together, our results provide new mechanistic insights into the tumor-suppressive functions of SMAD7 in hepatocarcinogenesis.

Presence of regulatory sequences within intron 4 of human and murine c-myb genes.

The molecular mechanisms that modulate c-myb mRNA transcription in hematopoietic cells appear to involve intron regulatory sequences. We have characterized the fourth of ten introns from both human and murine c-myb genes in regard to nucleotide sequence and specific protein binding. For this approach complete genomic c-myb intron 4 fragments were isolated from mouse and human DNA using PCR amplification with flanking exon-primers derived from the mouse gene. Comparison of the obtained sequences revealed strong homology between the two species. Using crude nuclear protein extracts from mouse and human myb expressing cells (70Z/3B; Molt4) and gel shift experiments we found specific protein interaction for both introns and to determine the protein binding site in detail, we performed DNase I footprinting. Our results indicate that the binding factor is absent in control cell lines without c-myb transcriptional activity, suggesting a possible positive regulatory function of the DNA-protein complex. To confirm these findings we introduced the human c-myb intron 4 DNA sequence into the EcoRI site of the pCAT-Promoter plasmid and transfected Molt4 cells with this chimeric construct. The transient expression studies revealed that intron 4 sequences possess enhancer activity. Thus, we have demonstrated that intron 4 sequences can be important for the regulation of c-myb proto-oncogene expression.

Gestational diabetes mellitus. Influence of race on disease prevalence and perinatal outcome in a U.S. population.

We explore whether racial differences in a United States population influence disease prevalence and perinatal outcome in gestational diabetes mellitus (GDM). The data presented are based on 3744 consecutive patients who underwent universal screening at 24-28 wk gestation; those with a 1-h plasma glucose greater than or equal to 7.2 mM underwent a 100-g 3-h oral glucose tolerance test (OGTT). The overall prevalence of GDM was 3.5 cases/100 with the standard O'Sullivan-Mahan diagnostic criteria derived for plasma, whereas use of the Carpenter-Coustan modification of the O'Sullivan-Mahan criteria yielded a prevalence of 5.5. The population was 39.1% white, 37.7% black, 19.8% Hispanic, and 3.4% Oriental/other. For those patients with a nondiagnostic test, mean plasma glucose at each time point of the OGTT was similar for all racial groups. Because of demographic and phenotypic heterogeneity between different racial groups, the influence of these different variables on the prevalence of GDM was tested by multiple logistic regression. Black and Hispanic race, maternal age, and percentage ideal body weight were found to have significant independent effects on the prevalence of GDM (P less than 0.05, 0.001, 0.001, and 0.001, respectively). The adjusted relative risk of GDM was significantly higher in black (1.81, 95% confidence interval [CI] 1.13-2.89, P less than 0.05) and Hispanic (2.45, 95% CI 1.48-4.04, P less than 0.001) patients compared with whites. The influence of race on infant birth weight was examined in the 92 patients with GDM controlled with diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Gestational diabetes mellitus. Heterogeneity of maternal age, weight, insulin secretion, HLA antigens, and islet cell antibodies and the impact of maternal metabolism on pancreatic B-cell and somatic development in the offspring.

We have examined gravida with gestational diabetes mellitus (GDM), as defined by the National Diabetes Data Group (Diabetes 1979; 28:1039), for phenotypic and genotypic heterogeneity. Fasting plasma glucose (FPG) at diagnosis was used for further stratification of GDM according to putative metabolic severity into class A1 (FPG less than 105 mg/dl [N = 129]), class A2 (FPG 105-129 mg/dl [N = 47]), and class B1 (FPG greater than or equal to 130 mg/dl [N = 23]). All GDM classes tended to be older and heavier than consecutive gravida with documented normal glucose tolerance (controls, N = 148). Subdivision into "lean" and "obese" indicated that plasma immunoreactive insulin (IRI) was greater after overnight fast in the obese of all groups except B1. However, absolute increases in IRI above fasting levels in response to glucose during OGTT were significantly enhanced by obesity only in class A2 gravida. Adjustment for the effects of age and weight by covariate analysis indicated that the IRI response to glycemic stimulation is usually attenuated in all forms of GDM. Mean values for increases in IRI above fasting values during the first 15 min and IRI increments relative to the increases in plasma glucose throughout the 180-min OGTT were below control values in all GDM groups and progressively so, i.e., A1 less than A2 less than B1. The absolute insulinopenia was not invariable; a small number of gravida from all GDM groups displayed well-preserved IRI responses to oral glucose. Genotypic evaluation of the GDM population disclosed an increased occurrence of "markers" known to be associated with type I diabetes mellitus.(ABSTRACT TRUNCATED AT 250 WORDS)

Tuberculosis in health care workers at a hospital with an outbreak of multidrug-resistant Mycobacterium tuberculosis.

OBJECTIVE: Investigate reports of tuberculosis in health care workers employed at a hospital with an outbreak of multidrug-resistant Mycobacterium tuberculosis. DESIGN: Case series of tuberculosis in health care workers, January 1, 1989, through May 31, 1992. Antimicrobial susceptibility testing and restriction fragment length polymorphism analysis of M tuberculosis isolates. Longitudinal analysis of cumulative tuberculin skin test surveillance data. Assessment of infection control. The patients consisted of 361 health care workers who had either serial tuberculin skin tests or tuberculosis. RESULTS: Six health care workers, the largest number linked to one multidrug-resistant tuberculosis outbreak, had disease due to M tuberculosis that matched the outbreak strain from hospitalized patients. The two who were seropositive for human immunodeficiency virus died, one of tuberculous meningitis and the other of multiple causes including tuberculosis. The estimated risk of a skin test conversion was positively associated with time and increased by a factor of 8.3 (1979 to 1992). In 1992 the annual risk for workers in the lowest exposure occupational group was 2.4%. In comparison, nurses and housekeepers had relative risks of 8.0 (95% confidence interval, 3.2 to 20.3) and 9.4 (95% confidence interval, 2.7 to 32.3), respectively. Laboratory workers had a relative risk of 4.2 (95% confidence interval, 1.1 to 15.5). Tuberculosis admissions increased, but the hospital had inadequate ventilation to isolate tuberculosis patients effectively. There were lapses in infection control practices. CONCLUSIONS: Health care workers who were exposed during a hospital outbreak of multidrug-resistant tuberculosis had occupationally acquired active disease. The human immunodeficiency virus-infected health care workers with tuberculosis had severe disease and died. The risk of skin test conversion increased during the study period, and higher exposure occupations had elevated risk. Effective infection control is essential to prevent the transmission of tuberculosis to health care workers.

Effect of pregnancy on renal function in patients with moderate-to-severe diabetic renal insufficiency.

OBJECTIVE: Previous studies of patients with diabetic nephropathy and mild renal impairment have suggested no determination in renal function as a result of pregnancy. The objective of this study was to determine whether pregnancy may permanently worsen renal function in women with diabetic nephropathy and moderate-to-severe renal insufficiency. RESEARCH DESIGN AND METHODS: Eleven patients were identified with diabetic nephropathy and moderate-to-severe renal dysfunction (creatinine [Cr] > or = 124 mumol/l [1.4 mg/dl]) at pregnancy onset by retrospective chart review. Alterations in glomerular filtration rate were estimated by using linear regression of the reciprocal of Cr over time. An equal number of nonpregnant premenopausal type 1 diabetic women with similar degrees of renal dysfunction served as a comparison group for nonpregnant rate of decline of renal function and potential contributing factors. RESULTS: Mean serum Cr rose from 159 mumol/l (1.8 mg/dl) prepregnancy to 221 mumol/l (2.5 mg/dl) in the third trimester. Renal function was stable in 27%, showed transient worsening in pregnancy in 27%, and demonstrated a permanent decline in 45%. Proteinuria increased in pregnancy in 79%. Exacerbation of hypertension or preeclampsia occurred in 73%. Seven patients progressed to dialysis 6-57 months postpartum, with 71% (five of seven) of these cases attributed to acceleration of disease during the pregnancy. Student's tests and repeated-measures analysis of variance support a pregnancy-induced acceleration in the rate of decline of renal function. CONCLUSIONS: In this series, patients with diabetic nephropathy and moderate-to-severe renal insufficiency were found to have a > 40% chance of accelerated progression of their disease as a result of pregnancy.

A rapid protocol for the purification of mitochondrial DNA suitable for studying restriction fragment length polymorphisms.

When analyzing mitochondrial DNA (mtDNA) from various normal and malignant human tissues, it became necessary to enhance mtDNA isolation for improved yields and quality. The method described here consists of rapid and simple-to-perform steps, avoiding complicated instrumentation. It was designed for preparation of undegraded mtDNA and is highly useful when limited amounts of tissues, cells and unique biopsies of tumors (fresh or frozen) are available. The resulting mtDNA is sufficiently pure for restriction analysis, subcloning, labeling and various types of hybridization. Using Sau3A and MspI, restriction analysis revealed new restriction-fragment length polymorphisms for Caucasians, independent of the DNA source, and hence excluding tissue-specific DNA modifications.

Cross-hybridization between the avian myeloblastosis oncogene and eukaryotic 28S ribosomal RNA.

In Northern blots, avian myeloblastosis (myb) oncogene probes (genomic or cDNA) cross-hybridize to the 28S rRNA band mimicking a myb-specific transcript. A misinterpretation of the hybridization data can be avoided by using an oligodeoxyribonucleotide probe.

Transforming growth factor beta signal transduction in hepatic stellate cells via Smad2/3 phosphorylation, a pathway that is abrogated during in vitro progression to myofibroblasts. TGFbeta signal transduction during transdifferentiation of hepatic stellate cells.

To current knowledge, transforming growth factor beta (TGFbeta) signaling is mandatory to establish liver fibrosis and various molecular interventions designed to affect the TGFbeta system were successfully used to inhibit fibrogenesis. Activated hepatic stellate cells (HSC), which are one important source of TGFbeta, are the major producers of extracellular matrix proteins in liver injury. We have previously shown that the TGFbeta response of this cell type is modulated during the transdifferentiation process. This work delineates the activation of TGFbeta downstream mediators, the Smads, in quiescent HSC and transdifferentiated myofibroblasts (MFB). The expression level of all Smads remained largely unchanged during this process. The response of HSC to TGFbeta, leading to, e.g., induction of alpha2 (I) collagen expression, is mediated by phosphorylation of Smad2 and Smad3 and subsequent nuclear translocation of a Smad containing complex. Neither TGFbeta-dependent nor endogenously phosphorylated Smad2/3 was detectable in comparable amounts in transdifferentiated MFB, indicating loss of TGFbeta sensitivity. Ectopic expression of Smad7 in HSC led to inhibition of Smad2 phosphorylation and abrogated TGFbeta response. In transdifferentiated MFB, expression of a constitutively active TGFbeta receptor I, but not treatment with TGFbeta1, resulted in transcriptional activation of a TGFbeta responsive promoter, thereby demonstrating completely restored TGFbeta signal transduction. Our data indicate that in contrast to a postulated mechanism of enduring autocrine TGFbeta signal transduction, early and late stages of HSC activation have to be distinguished, which is of importance for antifibrotic therapies.

Nonradioactive southwestern analysis using chemiluminescent detection.

Replacing radioactively labeled probes by nonradioactive ones and detection by chemiluminescence instead of colorimetry allows a nonhazardous handling and offers the possibility of easily reprobing filters in Southwestern analysis. Using the described procedure, we were able to determine the molecular weight of DNA-binding proteins and achieve a high signal-to-noise ratio.

c-myb intron I protein binding and association with transcriptional activity in leukemic cells.

Specific binding of nuclear proteins to the region of transcriptional attenuation has been shown to modulate the expression of c-myb, a nuclear proto-oncogene preferentially expressed in lympho-hematopoietic cells. Here, it plays an important role in processes of differentiation and proliferation. The mechanism that regulates c-myb expression is not yet fully understood. The block of transcriptional elongation which has been mapped to a 1 kb region within murine intron 1 may represent one regulatory pathway. The DNA sequences containing the transcriptional pause site are well conserved between murine and human species, thus Implying similar transcription-control strategies. We compared the binding potential of nuclear extracts (from human fibroblasts and MOLT4 as well as murine NIH3T3- and 70Z/3B- cell lines) to oligonucleotide sequences previously shown to be target binding sites in the murine system. One complex containing a 70 D protein was found to be associated specifically with transcriptionally active leukemia cells. We performed transient expression studies with a CAT reporter construct containing this putative enhancer sequence and yielded significant CAT activity. We identified further a putative 20 kD repressor protein in transcriptionally silent cells and demonstrated that c-Jun is part of an ubiquitously present complex. Our results confirm the participation of intron 1 in transcriptional regulation of the c-myb gene (in mouse and human) and implicate multiple and complex regulatory mechanisms of activation during myelomonocytic differentiation and leukemic cell growth control.