Fusion with E2A converts the Pbx1 homeodomain protein into a constitutive transcriptional activator in human leukemias carrying the t(1;19) translocation.

E2A-PBX1 is a chimeric gene formed by the t(1;19)(q23;p13.3) chromosomal translocation of pediatric pre-B-cell leukemia. The E2A-Pbx1 fusion protein contains sequences encoding the transactivation domain of E2A joined to a majority of the Pbx1 protein, which contains a novel homeodomain. Earlier, we found that expression of E2A-Pbx1 causes malignant transformation of NIH 3T3 fibroblasts and induces myeloid leukemia in mice. Here we demonstrate that the homeodomains encoded by PBX1, as well as by the highly related PBX2 and PBX3 genes, bind the DNA sequence ATCAATCAA. E2A-Pbx1 strongly activates transcription in vivo through this motif, while Pbx1 does not. This finding suggests that E2A-Pbx1 transforms cells by constitutively activating transcription of genes regulated by Pbx1 or by other members of the Pbx protein family.

Oncogenic activation of the Lck protein accompanies translocation of the LCK gene in the human HSB2 T-cell leukemia.

The tyrosine protein kinase p56lck transduces signals important for antigen-induced T-cell activation. In transgenic mice, p56lck is oncogenic when overexpressed or expressed as a mutant, catalytically activated enzyme. In humans, the LCK gene is located at the breakpoint of the t(1;7)(p34;q34) chromosomal translocation. This translocation positions the beta T-cell receptor constant region enhancer upstream of the LCK gene without interrupting the LCK coding sequences, and a translocation of this sort occurs in both the HSB2 and the SUP-T-12 T-cell lines. We have found that, although the level of the p56lck protein in HSB2 cells is elevated approximately 2-fold in comparison with that in normal T-cell lines, total cellular tyrosine protein phosphorylation is elevated approximately 10-fold. Increased levels of phosphotyrosine in HSB2 cells resulted from mutations in the LCK gene that activated its function as a phosphotransferase and converted it into a dominant transforming oncogene. The oncogenic p56lck in HSB2 cells contained one amino acid substitution within the CD4/CD8-binding domain, two substitutions in the kinase domain, and an insertion of Gln-Lys-Pro (QKP) between the SH2 and kinase domains. In NIH 3T3 fibroblasts, three of these mutations cooperated to produce the fully oncogenic form of this p56lck variant. These results suggest that mutation of LCK may contribute to some human T-cell leukemias.

Both Pbx1 and E2A-Pbx1 bind the DNA motif ATCAATCAA cooperatively with the products of multiple murine Hox genes, some of which are themselves oncogenes.

E2A-PBX1 is the oncogene produced at the t(1;19) chromosomal breakpoint of pediatric pre-B-cell leukemia. Expression of E2A-Pbx1 induces fibroblast transformation and myeloid and T-cell leukemia in mice and arrests differentiation of granulocyte macrophage colony-stimulating factor-dependent myeloblasts in cultured marrow. Recently, the Drosophila melanogaster protein Exd, which is highly related to Pbx1, was shown to bind DNA cooperatively with the Drosophila homeodomain proteins Ubx and Abd-A. Here, we demonstrate that the normal Pbx1 homeodomain protein, as well as its oncogenic derivative, E2A-Pbx1, binds the DNA sequence ATCAATCAA cooperatively with the murine Hox-A5, Hox-B7, Hox-B8, and Hox-C8 homeodomain proteins, which are themselves known oncoproteins, as well as with the Hox-D4 homeodomain protein. Cooperative binding to ATCAATCAA required the homeodomain-dependent DNA-binding activities of both Pbx1 and the Hox partner. In cotransfection assays, Hox-B8 suppressed transactivation by E2A-Pbx1. These results suggest that (i) Pbx1 may participate in the normal regulation of Hox target gene transcription in vivo and therein contribute to aspects of anterior-posterior patterning and structural development in vertebrates, (ii) that E2A-Pbx1 could abrogate normal differentiation by altering the transcriptional regulation of Hox target genes in conjunction with Hox proteins, and (iii) that the oncogenic mechanism of certain Hox proteins may require their physical interaction with Pbx1 as a cooperating, DNA-binding partner.

Oncoprotein E2A-Pbx1 immortalizes a myeloid progenitor in primary marrow cultures without abrogating its factor-dependence.

E2A-PBX1 is a chimeric homeobox oncogene formed by the t(1;19) translocation of human pre-B cell acute lymphoblastic leukemia (ALL). In a previous study, we found that retroviral expression of E2A-Pbx1 in the marrow of reconstituted mice induced the formation of acute myeloid leukemia (AML) in vivo. Here, we report that E2A-Pbx1 can also immortalize myeloid progenitors in vitro, and that the outgrowth of immortalized myeloblasts is evident only in the presence of the myeloid lymphokine, granulocyte-macrophage colony stimulating factor (GM-CSF). When cultured in the presence of GM-CSF, responsive myeloblasts from normal marrow exhibit concurrent proliferation and differentiation, and undergo terminal differentiation into non-mitotic neutrophils and macrophages within 4 weeks. Infection of identical cultures with a retrovirus encoding E2A-Pbx1 produces a rapid outgrowth of myeloid progenitors that express high levels of E2A-Pbx1 protein. A small fraction of myeloblasts in each population exhibited limited differentiation to neutrophils, and all populations of myeloblasts retained a strict dependence on GM-CSF for both survival and proliferation. This data suggests that the function of E2A-Pbx1 in leukemias is to strongly retard differentiation without affecting growth-factor dependence.

DNA-binding by oncoprotein E2a-Pbx1 is important for blocking differentiation but dispensable for fibroblast transformation.

The t(1;19) chromosomal translocation of pediatric pre-B cell lymphoblastic leukemia produces the E2A-PBX1 oncogene, which can transform fibroblasts, induce acute myeloid leukemia and T cell lymphomas in mice, and immortalize factor-dependent myeloid progenitors in cultured marrow. The homeodomain of Pbx1 binds ATCAATCAA, and while Pbx1 does not activate transcription through this motif, E2A-Pbx1 induces constitutive transactivation. Here, we investigate whether DNA-binding by Pbx1 or transcriptional activation by E2A are essential for the transforming abilities of E2A-Pbx1. Elimination of DNA-binding in E2A-Pbx1 by point mutations in the Pbx1 homeodomain or by large deletions that removed the Pbx1 homeodomain and carboxyl terminus did not alter ability of E2A-Pbx1 to induce focus-formation in fibroblast, even though these mutations completely eliminated its ability to activate transcription through the PRS. These same DNA-binding mutations, however, severely impaired or eliminated the ability of E2A-Pbx1 to immortalize factor-dependent myeloid progenitors in marrow cultures. Elimination of the first transcriptional activation domain of E2A abolished both fibroblast and myeloid transforming activities while elimination of the second altered neither of these activities. We conclude that DNA-binding is important for the ability of E2A-Pbx1 to disrupt differentiation, as evidenced in myeloblast immortalization, but dispensable for its ability to induce focus-formation, and that the aminoterminal domain of E2A, which strongly activates transcription, is essential for both transforming activities.

Mossy fiber projections from the cuneate nucleus to the cochlear nucleus in the rat.

A reciprocal connection is known to exist between the cuneate nucleus, which is a first-order somatosensory nucleus, and the cochlear nucleus, which is a first-order auditory nucleus. We continued this line of study by investigating the fiber endings of this projection in the cochlear nucleus of rats using the neuronal tracer Phaseolus vulgaris leucoagglutinin in combination with ultrastructural and immunocytochemical analyses. In the cochlear nucleus, mossy fiber terminals had been described and named for their morphologic similarity to those in the cerebellum, but their origins had not been discovered. In the present study, we determined that the axonal projections from the cuneate region gave rise to mossy fiber terminals in the granule cell regions of the ipsilateral cochlear nucleus. The cuneate mossy fibers appear to be excitatory in nature, because they are filled with round synaptic vesicles, they make asymmetric synapses with postsynaptic targets, and they are labeled with an antibody to glutamate. The postsynaptic targets of the mossy fibers include dendrites of granule cells. This projection onto the granule cell interneuron circuit of the cochlear nucleus indicates that somatosensory cues are intimately involved with information processing at this early stage of the auditory system.

Inositol 1,4,5-trisphosphate receptors: immunocytochemical localization in the dorsal cochlear nucleus.

In the cochlear nucleus of mammals, the relatively homogeneous responses of auditory nerve fibers are transformed into a variety of different response patterns by the different classes of resident neurons. The spectrum of these responses is hypothesized to depend on the types and distribution of receptors, ion channels, G proteins, and second messengers that form the signaling capabilities in each cell class. In the present study, we examined the immunocytochemical distribution of the inositol 1,4,5-triphosphate (IP3) receptor in the dorsal cochlear nucleus to better understand how this second messenger might be involved in shaping the neural signals evoked by sound. Affinity-purified polyclonal antibodies directed against the IP3 receptor labeled a homogeneous population of neurons in the dorsal cochlear nucleus of rats, guinea pigs, mustache bats, cats, New World owl monkeys, rhesus monkeys, and humans. These cells were all darkly immunostained except in the human where the labeling was less intense. Immunoblots of dorsal cochlear nucleus tissue from the rat revealed a single band of protein of molecular weight approximately 260 kD, which is the same size as the purified receptor, indicating that our antibodies reacted specifically with the IP3 receptor. These immunolabeled neurons were identified as cartwheel cells on the basis of shared characteristics across species, including cell body size and distribution, the presence of a highly invaginated nucleus, and a well-developed system of cisternae. Reaction product was localized along the membranes of rough and smooth endoplasmic reticulum, subsurface cisternae, and the nuclear envelope. This label was distributed throughout the cartwheel cell body and dendritic shafts but not within dendritic spines, axons, or axons terminals. The regular pattern of immunolabeling across mammals suggests that IP3 and cartwheel cells are conserved in evolution and that both play an important but as yet unknown role in hearing.

Neuronal organization of the cochlear nuclei in alligator lizards: a light and electron microscopic investigation.

The organization of neurons and fibers in the cochlear nuclei of the alligator lizard (Gerrhonotus multicarinatus) was examined with light and electron microscopy. In this species, much is known about the anatomy and physiology of the inner ear including the cochlear nerve, but little is known about the synaptic connections of cochlear fibers on second-order neurons. These data will help to develop general principles addressing the cellular organization of the vertebrate auditory system. Subdivisions of the cochlear nuclei were defined on the basis of their histologic appearance and neuronal composition. Neuron classes were proposed from their light microscopic and ultrastructural features. Nucleus magnocellularis medialis consists of a homogeneous population of neurons called "lesser ovoid" cells. Nucleus magnocellularis lateralis consists of "greater ovoid" and "small" cells. Nucleus angularis lateralis consists of "spindle" cells. Lastly, nucleus angularis medialis contains a population of large neurons called "duckhead" and "multipolar" cells, and a population of smaller neurons called "bulb" and "agranular" cells. These neuron populations are differentially innervated by tectorial and free-standing cochlear fibers that are associated with separate frequency ranges. All neuronal populations except agranular cells were observed to receive synaptic input from cochlear nerve fibers. In nucleus magnocellularis medialis and nucleus angularis medialis, primary afferents form both chemical and electrical synapses with resident neurons. These observations imply that acoustic information is synaptically processed in fundamentally distinct ways in the cochlear nuclei of alligator lizards and distributed along separate neural circuits. Thus, the characteristic structural and functional dichotomy of the alligator lizard inner ear is extended to central auditory pathways by way of cochlear nerve projections.

Frequency organization of the dorsal cochlear nucleus in cats.

Sensory epithelia are often spatially reiterated throughout their representation in the central nervous system. Differential expression of this representation can reveal specializations of the organism's behavioral repertoire. For example, the nature of the central representation of sound frequency in the auditory system has provided important clues in understanding ecological pressures for acoustic processing. In this context, we used electrophysiological techniques to map the frequency organization of the dorsal cochlear nucleus in nine cats. Frequency responses were sampled in increments of 100-200 microns along electrode tracks that entered the dorsomedial border of the nucleus and exited at the ventrolateral border. Electrode tracks were oriented parallel to the long (or strial) axis of the nucleus so that each penetration sampled neural responses for most of the cat's audible frequencies and remained in or near the pyramidal cell layer for several millimeters. Nearly identical distance versus frequency relationships were obtained for different rostral-caudal locations within the same cat as well as for different cats. Frequency responses systematically decreased from above 50 kHz at the most dorsomedial locations in the nucleus to below 1 kHz in the most ventrolateral regions. The rate of frequency change was roughly three times greater in high frequency regions than in low frequency regions. In addition, the highest pyramidal cell density and longest rostral-caudal axis was observed for the middle third of the dorsal-ventral axis of the nucleus. As a result, roughly half of all pyramidal cells responded to frequencies between 8-30 kHz. The representation of neural tissue for these frequencies may be related to the importance of spectral cues in sound locations.

Physiology and morphology of complex spiking neurons in the guinea pig dorsal cochlear nucleus.

Intracellular recordings from the dorsal cochlear nucleus have identified cells with both simple and complex action potential waveforms. We investigated the hypothesis that cartwheel cells are a specific cell type that generates complex action potentials, based on their analogous anatomical, developmental, and biochemical similarities to cerebellar Purkinje cells, which are known to discharge complex action potentials. Intracellular recordings were made from a brain slice preparation of the guinea pig dorsal cochlear nucleus. A subpopulation of cells discharged a series of two or three action potentials riding on a slow depolarization as an all-or-none event; this discharge pattern is called a complex spike or burst. These cells also exhibited anodal break bursts, anomalous rectification, subthreshold inward rectification, and frequent inhibitory postsynaptic potentials (IPSPs). Seven complex-spiking cells were stained with intracellular dyes and subsequently identified as cartwheel neurons. In contrast, six identified simple-spiking cells recorded in concurrent experiments were pyramidal cells. The cartwheel cell bodies reside in the lower part of layer 1 and the upper part of layer 2 of the nucleus. The cells are characterized by spiny dendrites penetrating the molecular layer, a lack of basal dendritic processes, and an axonal plexus invading layers 2 and 3, and the inner regions of layer 1. The cartwheel cell axons made putative synaptic contacts at the light microscopic level with pyramidal cells and small cells, including stellate cells, granule cells, and other cartwheel cells in layers 1 and 2. The axonal plexus of individual cartwheel cells suggests that they can inhibit cells receiving input from either the same or adjacent parallel fibers and that this inhibition is distributed along the isofrequency contours of the nucleus.

Immunocytochemical localization of the mGluR1 alpha metabotropic glutamate receptor in the dorsal cochlear nucleus.

We demonstrate that the metabotropic glutamate receptor mGluR1 alpha is enriched in two interneuron cell populations in the dorsal division of the cochlear nucleus. Electron microscopic analysis confirms that mGluR1 alpha immunoreactivity is concentrated in the dendritic spines of cartwheel cells and in dendrites of the recently described unipolar brush cells. The cartwheel cells, which have many similarities to the Purkinje cells of the cerebellum, participate in a local neuronal circuit that modulates the output of the dorsal cochlear nucleus. Immunostained unipolar brush cells were observed in granule cell regions of the cochlear nucleus and the vestibulocerebellum. The presence of analogous cell types with similar patterns of immunolabeling in the cerebellum and in the dorsal cochlear nucleus suggests that a shared but as yet unknown mode of processing may occur in both structures.

Effects of S-methyl glutathione, S-methyl cysteine, and the concentration of oxidized glutathione on transendothelial fluid transport.

Endothelial perfusions of preswollen rabbit corneal preparations with S-methyl glutathione at levels of 2 x 10(-4)M and 10(-3)M achieve about 70% to 100%, respectively, of the stromal thinning attained with 10(-4)M oxidized glutathione (GSSG). S-methyl cysteine at 10(-4)M is without effect and, at 10(-4)M, inhibits the fluid pump. The results are interpreted to signify that thiol-disulfide exchanges with GSSG probably are not an obligatory part of the endothelial pump mechanism. Contrary to the frequently inhibitory effect of high levels of GSSG on various enzyme systems, increasing the concentration of GSSG from 10(-4)M to 10(-2)M stimulates the first hour rate of stromal thinning 33%. Neither the total amount nor the duration of thinning, however, is significantly affected.

Shear strength of composite bonded to Er:YAG laser-prepared dentin.

An Er:YAG laser coupled with a cooling stream of water effectively removes dental hard tissues. However, before such a system can be deemed clinically viable, some safety and efficacy issues must be addressed. We compared the bonding of composite to dentin following the preparation of the dentinal surface with either an Er:YAG laser (lambda = 2.94 microns) or a standard dental bur and with and without a subsequent acid-etching treatment. The crowns of extracted human molars were removed, revealing the underlying dentin. We removed an additional thickness of material with either a dental handpiece or an Er:YAG laser (350 mJ/pulse at 6 Hz) by raster-scanning the samples under a fixed handpiece or laser. Comparable surface roughnesses were obtained. Several samples from each group received an acid-conditioning treatment. A cylinder of composite was bonded onto the prepared surfaces. The dentin-composite bond was then shear-stressed to failure on a universal testing apparatus. The results indicate that laser-irradiated samples had improved bond strengths compared with acid-etched and handpiece controls. SEM photographs of the surfaces show exposed tubules following the laser treatment: tubules could also be exposed with acid etching. We conclude that Er:YAG laser preparation of dentin leaves a suitable surface for strong bonding or an applied composite material.

GC-EC analysis of polybrominated biphenyl constituents of Firemaster FF-1 using tetrabromobiphenyl as an internal standard.

The polybrominated biphenyl (PBB) constituents of Firemaster FF-1 were determined using gas chromatography-electron capture (GC-EC). The absolute and relative retention times of each of eight major constituents were obtained. It was shown that 2,2',5,5'-tetrabromobiphenyl was a suitable internal standard for the quantitative analysis of the PBBs in Firemaster FF-1 in mammalian tissues and fluids.

Sustained compression and healing of chronic venous ulcers.

STUDY OBJECTIVE: Comparison of four layer bandage system with traditional adhesive plaster bandaging in terms of (a) compression achieved and (b) healing of venous ulcers. DESIGN: Part of larger randomised trial of five different dressings. SETTING: Outpatient venous ulcer clinic in university hospital. PATIENTS: (a) Pressure exerted by both bandage systems was measured in the same 20 patients. (b) Healing with the four layer bandage was assessed in 148 legs in 126 consecutive patients (mean age 71 (SE 2); range 30-96) with chronic venous ulcers that had resisted treatment with traditional bandaging for a mean of 27.2 (SE 8) months. INTERVENTIONS: (a) Four layer bandage system or traditional adhesive plaster bandaging for pressure studies; (b) four layer bandaging applied weekly for studies of healing. END POINTS: (a) Comparison of pressures achieved at the ankle for up to one week; (b) complete healing within 12 weeks. MEASUREMENTS AND MAIN RESULTS: (a) Four layer bandage produced higher initial pressures at the ankle of 42.5 (SE 1) mm Hg compared with 29.8 (1.8) for the adhesive plaster (p less than 0.001; 95% confidence interval 18.5 to 6.9). Pressure was maintained for one week with the four layer bandage but fell to 10.4 (3.5) mm Hg at 24 hours with adhesive plaster bandaging. (b) After weekly bandaging with the four layer bandage 110 of 48 venous ulcers had healed completely within 12 (mean 6.3 (0.4)) weeks. CONCLUSION: Sustained compression of over 40 mm Hg achieved with a multilayer bandage results in rapid healing of chronic venous ulcers that have failed to heal in many months of compression at lower pressures with more conventional bandages.

A method for assessing the outcome of acute primary care.

Some 1,700 acute care episodes were studied to assess the outcomes in terms of the extent to which patients regained their usual functional status. Involving active follow-up of each patient, the study serves as a prototype for measuring several components of quality of care including actual outcomes, patient expectation of outcome, physician expectation of outcome, and patient satisfaction with outcome and care. Because this study was conducted in a family practice residency training setting, we hope that it will serve as a model of how such information may be used to increase residents' sensitivity to the course of illness commonly seen in primary care, and to encourage the residents to set expectations for the care they give.

Differences in the outcomes of acute episodes of care provided by various types of family practitioners.

This study was designed to compare the outcomes achieved in a series of acute care episodes by different levels of family practice providers working in the clinic setting. The study utilizes a method which depends upon the provider to estimate level of function expected and earliest date of recovery for each episode. When the patients are viewed as a single group, those patients treated by the medex appear to fare considerably better and those seen by a faculty member do worse; however, when each functional status group is examined separately, only the asymptomatic but clinically ill patients (45 cases) show a statistically significant difference in outcomes among the providers, with the medex having good results and the faculty poor results.

Something's missing from the video: An alternative instructional approach to "An Ounce of Prevention".

CONCLUSION: The question of why there is a need to be aware of the theoretical principles of prevention and prevention programs raises several responses. First, there appears to be higher incidents of inadequate responses to the transitions of life, normative and otherwise, as indicated by the scope of the projects presented in the video. Instead of alleviating the frustration and stress associated with change, and thereby reducing the incidence of disorders within the community, inadequate coping mechanisms exacerbate the impact of the change on the individual. Therefore, programs which develop social skills that alleviate the impact of stressors on the individual will reduce the overall impact of disorders in the community.Second, there has been a general decrease in the number of services which adequately address health related needs on the federal, state, local, and private levels. For those who are in need of service, this lack of availability compounded with the lack of community, relegates the individual to a life of isolation. A core theme that emerged from the video was when individuals are isolated from the community there is a higher occurrence of poor coping mechanisms in response to stressors.Third, as general health care moves towards the 21st century, managed health care systems are becoming the mode of accepted treatment. In an attempt to keep the cost of health care reasonable, many facilities are encouraging their participants to seek resolution to problems before they become detrimental to the overall well being of the participants.Lastly, as professional in the fields of psychology, sociology, and public policy, it is imperative that a thorough understanding of the ideological principles of prevention and prevention programs are presented to ensure the development of programs which adequately meet the needs of future societies.

Recent rural health research.

Recent rural health research may be examined in two ways: needs and solutions. A definition of needs requires an evaluation of the social factors that affect the expectations and the behavior of both the provider and the consumer. Three types of solutions should be considered: the appropriate utilization of manpower, including the efforts to influence physician location and specialty distribution, new health practitioners, and team approaches; the new technology for transportation and communication; and the organization of new delivery systems. Two areas of rural health research that need more attention are program evaluation and financial planning.

Predicting the outcome of primary care.

Each physician's ability to treat disease is limited by his/her ability to discriminate among patients on the basis of risk. The relationship of physician expectation of outcome (prognosis) to actual outcome and to cost were determined for 1,757 patients seeking primary care. Outcome was measured by a seven-level functional-status scale; patients who returned to their usual level of function after an acute illness were defined as having good outcomes. Although 24 per cent of patients experienced bad outcomes, physicians had anticipated only 6 per cent. Physician's predictions of bad outcomes had a sensitivity of 13.6 per cent and a specificity of 96.9 per cent. Patients with bad outcomes had slightly higher laboratory costs than did patients with good outcomes, but a much larger increase was seen in laboratory, office and total costs for all patients for whom physicians expected bad outcomes, regardless of the actual results. A feedback loop is recommended to provide a better perspective on outcome and eventually to improve the efficiency and cost benefit of the medical decision-making process.