Disrupted differentiation and oncogenic transformation of lymphoid progenitors in E2A-HLF transgenic mice.

The hepatic leukemia factor (HLF) gene codes for a basic region-leucine zipper (bZIP) protein that is disrupted by chromosomal translocations in a subset of pediatric acute lymphoblastic leukemias. HLF undergoes fusions with the E2A gene, resulting in chimeric E2a-Hlf proteins containing the E2a transactivation domains and the Hlf bZIP DNA binding and dimerization motifs. To investigate the in vivo role of this chimeric bZIP protein in oncogenic transformation, its expression was directed to the lymphoid compartments of transgenic mice. Within the thymus, E2a-Hlf induced profound hypoplasia, premature involution, and progressive accumulation of a T-lineage precursor population arrested at an early stage of maturation. In the spleen, mature T cells were present but in reduced numbers, and they lacked expression of the transgene, suggesting further that E2a-Hlf expression was incompatible with T-cell differentiation. In contrast, mature splenic B cells expressed E2a-Hlf but at lower levels and without apparent adverse or beneficial effects on their survival. Approximately 60% of E2A-HLF mice developed lymphoid malignancies with a mean latency of 10 months. Tumors were monoclonal, consistent with a requirement for secondary genetic events, and displayed phenotypes of either mid-thymocytes or, rarely, B-cell progenitors. We conclude that E2a-Hlf disrupts the differentiation of T-lymphoid progenitors in vivo, leading to profound postnatal thymic depletion and rendering B- and T-cell progenitors susceptible to malignant transformation.

Functional determination of oxytocin affinity in near-term pregnant rat myometrium: effect of chronic hypoxia.

We designed the present study to determine: (1) if phenoxybenzamine can be used as an irreversible blocker for oxytocin receptors, and as such to determine oxytocin affinity, (2) if prolonged hypoxic exposure alters oxytocin receptor coupling efficacy of oxytocin receptors to post-receptor mediated mechanisms in the rat myometrium. Rats were exposed to room air (control), or to continuous hypoxia (10.5% O2) from day 19 through day 21 (2-day exposure). On day 21, one uterine horn was removed and used for in vitro study of myometrial contractile responses to oxytocin, while the other was used for oxytocin receptor analysis. In normoxic tissues, phenoxybenzamine (20 microM) decreased the maximum contractile response (EMAX) to oxytocin (155+/-17 vs. 66+/-19 g s/cm2) and oxytocin binding sites (BMAX: 253+/-35 vs. 114.9+/-21.3 fmol/mg protein). A similar degree of reduction in EMAX and BMAX were observed in hypoxic tissues. The oxytocin dissociation constant (KA) in the normoxic rat was 2.8+/-0.7 nM, which was not different from the chronic hypoxic rat (3.3+/-0.9 nM). Analysis of receptor occupancy-response curves indicated no oxytocin receptor reserve in both normoxic and hypoxic myometrium. However, for a given fraction of the total oxytocin receptors occupied, hypoxic tissue elicited a lower contractile response to oxytocin. We conclude that: (1) phenoxybenzamine is a useful tool to functionally study oxytocin receptor kinetics, (2) prolonged hypoxic exposure does not affect the oxytocin affinity, (3) no spare receptors for oxytocin are present in the rat myometrium, and (4) prolonged exposure to hypoxia decreases oxytocin receptor-effector coupling efficiency in rat myometrium.

Suppression of myometrial contractile responses to oxytocin after different durations of chronic hypoxia in the near-term pregnant rat.

OBJECTIVE: In light of our previous finding that chronic hypoxia decreases the myometrial contractile response to oxytocin in the near-term pregnant rat, we designed the current study (1) to investigate the effect of duration of hypoxic exposure on the contractile response to oxytocin and oxytocin binding sites and (2) to examine the effect of prolonged hypoxia on the contractile response to aluminum fluoride. STUDY DESIGN: Rats were exposed to room air (control) or to continuous hypoxia (10.5% oxygen) from day 19 through day 21 (48-hour exposure), from day 20 through day 21 (24-hour exposure), or midday 20 through day 21 of gestation (12-hour exposure). On day 21 the uterine horns were used for oxytocin receptor analysis and for in vitro study of myometrial contractile responses to cumulative doses of oxytocin (10(-10) to 10(-6) mol/L) or aluminum fluoride (0.5 to 4.0 mmol/L sodium fluoride in 10 mumol/L aluminum chloride). RESULTS: The maximal contractile tensions for the control and 12-hour exposure showed no difference. In contrast, 24-hour hypoxic exposure resulted in a reduction of the maximal contractile tension from 143 +/- 11 (control) to 116 +/- 7 gm x sec/cm2. By 48 hours the maximal contractile tension was reduced even further, to 44 +/- 13 gm x sec/cm2. Oxytocin binding sites followed a similar trend with values changing from 256.9 +/- 34.9 for control to 122.9 +/- 26.1 and 84.9 +/- 21.3 fmol/mg protein for the 24- and 48-hour exposure groups, respectively (p < 0.01, analysis of variance), with no change in the 12-hour group. The contractile responses to aluminum fluoride were not altered. CONCLUSIONS: The suppression in the myometrial contractile response to oxytocin and oxytocin binding sites depends on the duration of hypoxic exposure. Chronic hypoxic exposure did not affect the myometrial response to aluminum fluoride.

Use of a cast spreader during reoperative sternotomy.

We report a modified use for an old tool: separation of the sternal edges during repeat sternotomy with a cast spreader. We have found that this technique improves our exposure, causes no trauma to bone, and minimizes the risk of damage to underlying mediastinal structures.

Effect of chronic hypoxia on myometrial responsiveness in the pregnant rat.

Mechanisms involving the timing of normal parturition are not well understood in most animal species. To gain a greater understanding of the mechanisms, we employed hypoxia to perturb the normal system of parturition. The present study was designed to investigate the effects of chronic hypoxia on myometrial contractility in the near-term pregnant rat. Rats were exposed to room air (control) or to continuous hypoxia (10.5% O2) either from experimental days 19 through 21 (2-day exposure) or from experimental days 15 through 21 (6-day exposure). On day 21, blood was collected for hormone assays, and the uterine horns were collected from each dam. One horn was snap-frozen in liquid nitrogen for oxytocin (OT) receptor analysis, and the other was used for in vitro assessment of myometrial contractile responses to cumulative doses of OT or arginine vasopressin (AVP). Hypoxic exposure resulted in approximately 60% reduction of the maximal myometrial contractile response to OT and a significant reduction in OT binding sites from 256.9 +/- 34.9 to 84.9 +/- 21.3 fmol/mg protein (P<0.01). In contrast, the contractile response to AVP was unaffected after exposure to chronic hypoxia (P> 0.05). Additionally, we observed no difference in the plasma concentrations of estrogen, progesterone, and corticosterone. We conclude that chronic hypoxia decreased the effectiveness of OT-specific contractile mechanisms, at least partially through a decrease in OT binding sites.

Human CD6 possesses a large, alternatively spliced cytoplasmic domain.

Human CD6 is a monomeric 105/130-kDa T cell surface glycoprotein that is involved in T cell activation. The apparent discrepancy between the size of the cytoplasmic domain in human (44 amino acids) and mouse (243 amino acids) CD6, led us to use reverse transcriptase-polymerase chain reaction of human peripheral blood lymphocyte mRNA to isolate cDNA clones that include the carboxyl-terminal coding region of human CD6. The nucleotide sequence of the longest human cDNA clone, CD6-PB1, predicts a protein of 668 amino acids with a 244-amino acid cytoplasmic domain similar in size to and possessing 71.5% amino acid sequence identity with the cytoplasmic domain of mouse CD6. This previously unrecognized 244-amino acid cytoplasmic domain does not have significant homology to any other known protein (except mouse CD6), but does possess two proline-rich motifs containing the SH3 domain-binding consensus sequence, a serine-threonine-rich motif repeated three times, three protein kinase C phosphorylation-site motifs, and 10 casein kinase-2 phosphorylation-site motifs. These sequences are likely to play a role in the ability of CD6-specific monoclonal antibodies to stimulate T cell proliferation. Full-length CD6 cDNA containing this cytoplasmic domain sequence encodes a monomeric 105/130-kDa protein that can be immunoprecipitated from the surface of transfected cells and comigrates upon SDS-PAGE with wild-type CD6 immunoprecipitated from PBL. We also isolated two alternatively spliced forms of human CD6 cDNA lacking sequences encoding membrane-proximal regions of the cytoplasmic domain which maintain the same reading frame as CD6-PB1. The short cytoplasmic domain of the previously reported human CD6-15 cDNA clone results from a deletion of a 20-bp segment through use of an alternative 3' splice site, resulting in a frame shift and premature termination of translation relative to the clones we have isolated. These data demonstrate that human CD6 possesses a large cytoplasmic domain containing sequence motifs that are likely to be involved in signal transduction upon stimulation of T cells through CD6 ligation.

Indomethacin treatment of human adult respiratory distress syndrome.

The purpose of this investigation was to evaluate the magnitude and duration of changes in lung function and oxygen transport in patients with adult respiratory distress syndrome (ARDS) receiving indomethacin. Ten patients with ARDS were randomized to receive intravenously either a single 50 mg dose of indomethacin or placebo. Comparing 1 hr postinfusion levels to baseline observations in the indomethacin group, PaO2 increased to 125 +/- 13 torr from 93 +/- 8 torr, PaO2/FIO2 ratio increased to 223 +/- 24 from 160 +/- 5, and Qs/Qt dropped to 0.20 +/- 0.03 from 0.27 +/- 0.03 (all P less than 0.05). These alterations in oxygenation gradually returned to baseline levels over the ensuing 8 hr. No such changes were noted in the placebo group.

Adenosine A1 receptor mediated protection of the globally ischemic isolated rat heart.

The purpose of this study was to determine if the cardioprotective effect of adenosine on the ischemic myocardium is mediated by interaction with specific adenosine receptor subtypes. Isolated rat hearts perfused at constant flow were subjected to global normothermic (37 degrees C) ischemia and the time to onset of ischemic contracture (TOIC) was used as a marker of myocardial ischemic injury. Hearts treated with adenosine and R-phenylisopropyladenosine (PIA), an adenosine A1 receptor agonist, exhibited a significantly greater TOIC than control hearts (18.60 +/- 0.40 and 16.64 +/- 1.15 min, respectively vs 9.12 +/- 0.66 min), whereas phenylaminoadenosine, an adenosine A2 receptor agonist, had no effect on TOIC (11.73 +/- 0.87 min). BW A1433U, an adenosine receptor antagonist, blocked the effects of adenosine and PIA on ischemic contracture time, and BW A1433U did not alter the ability of nifedipine or propranolol to delay the onset of ischemic contracture, thus indicating the specificity of this compound for the adenosine receptor. PIA-treated hearts exhibited significantly greater ATP levels throughout the ischemic period compared to control hearts, whereas hearts treated with BW A1433U showed a rapid decline in ATP content. These results suggest that the beneficial effects of adenosine on the ischemic myocardium are mediated by interaction with adenosine A1 receptors, and that endogenously formed adenosine plays a role in attenuating myocardial ischemic damage.