Regulation of erythropoietin production in a human hepatoblastoma cell line

Production of immuno and biologically active erythropoietin was documented to occur in the human hepatoblastoma cell line HepG-2. The expression of the erythropoietin gene was further verified by Northern blot analysis using a single stranded RNA probe. In vitro studies showed that erythropoietin production by these cells was not stimulated by hypoxia or cobalt chloride, but was related to the proliferative activity of the cells in culture. In addition it was found that the secretion of erythropoietin was almost completely abrogated by tunicamycin, an inhibitor of N-linked glycosylation. This effect of tunicamycin was also observed in a permanently transfected cell line that secretes erythropoietin in large quantities.     

An alternative extrinsic pathway of human blood coagulation

To study the interrelationships of the major human coagulation pathways, factor X activation in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin were added to plasma samples containing 3H-labeled factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin the rate of factor X activation in factor VIII or factor IX deficient plasma was only 10% of the activation rate seen for normal or factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, restored normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these experiments, it is inferred that normal activation of factor X in plasma due to dilute thromboplastin requires factors VII, IX and VIII. An alternative extrinsic pathway that involves factors VII, IX, and VIII may be a major physiologic extrinsic pathway, and this pathway may help to explain the clinical observations of bleeding diatheses in patients deficient in factors IX or VIII.     

Clinical features and outcome of T-cell acute lymphoblastic leukemia in childhood with respect to alterations at the TAL1 locus: a Pediatric Oncology Group study

Alteration of the TAL1 locus is the most common nonrandom genetic defect in childhood T-cell acute lymphoblastic leukemia (T-ALL). To determine if rearrangements of the TAL1 proto-oncogene confer a distinct leukemic phenotype, we studied leukemic peripheral blood or bone marrow samples from 182 children with newly diagnosed T-ALL enrolled on Pediatric Oncology Group treatment protocols. Forty-eight (26%) of the samples had a local rearrangement of the TAL1 locus. Demographic and clinical features were compared for patient subgroups with and without TAL1 rearrangements. The only clinical correlates that were significantly associated with TAL1 gene rearrangements were higher white blood cell count (P = .017) and higher hemoglobin (P = .007) at diagnosis. Immunophenotypically, samples with altered TAL1 were more likely to be CD2+ (P = .001) and lack CD10 (cALLa) expression (P = .007) than those without the rearrangement. There was a trend toward improved event-free survival (EFS) in patients with TAL1 rearrangements (4-year EFS was 44% +/- 7% for patients without the rearrangements v 59% +/- 11% for those with rearrangements), but the difference was not significant (P = .34). The role of TAL1 in leukemogenesis has yet to be clearly defined, and the prognostic significance of TAL1 gene rearrangements in T-ALL deserves further study.     

Factor V deficiency in Philadelphia-positive chronic myelogenous leukemia

Factor V deficiency has been identified in 8 of 8 patients 7--20 yr of age, with Philadelphia-positive (Ph1+) chronic myelogenous leukemia (CML). In these 8 patients, factor V deficiency was not due to hepatic dysfunction, factor V inhibitors, or disseminated intravascular coagulation. In 3 patients, factor V activity rose 10%--12% (0.10--0.12 U/ml) after the infusion of 28--31 ml/kg body weight of fresh frozen plasma (FFP). The rise persisted less than 14 hr. The mean measured postinfusion rise in factor V was 18% of the expected rise calculated from the volume of FFP infused in the patients' plasma volume. In 4 patients, a small transient rise in factor V activity occurred after splenectomy or plateletpheresis. Factor V deficiency was completely corrected after a marked reduction in bone marrow cellularity in 2 patients with Ph1+ CML treated with extensive chemotherapy, total body irradiation, and bone marrow transplantation. Factor V deficiency was retrospectively observed in 6 of 20 patients, ages 20--80 yr, with Ph1+ CML and 3 of 6 patients with other myeloproliferative disorders. The factor V deficiency appears to be associated with the large myeloid- megakaryocytic cell mass characteristic of CML and other myeloproliferative disorders.     

Abnormal intracellular calcium homeostasis in sympathetic neurons from young prehypertensive rats

Hypertension is associated with cardiac noradrenergic hyperactivity, although it is not clear whether this precedes or follows the development of hypertension itself. We hypothesized that Ca(2+) homeostasis in postganglionic sympathetic neurons is impaired in spontaneously hypertensive rats (SHRs) and may occur before the development of hypertension. The depolarization-induced rise in intracellular free calcium concentration ([Ca(2+)](i); measured using fura-2-acetoxymethyl ester) was significantly larger in cultured sympathetic neurons from prehypertensive SHRs than in age matched normotensive Wistar-Kyoto rats. The decay of the [Ca(2+)](i) transient was also faster in SHRs. The endoplasmic reticulum Ca(2+) content and caffeine-induced [Ca(2+)](i) amplitude were significantly greater in the young SHRs. Lower protein levels of phospholamban and more copies of ryanodine receptor mRNA were also observed in the young SHRs. Depleting the endoplasmic reticulum Ca(2+) store did not alter the difference of the evoked [Ca(2+)](i) transient and decay time between young SHRs and Wistar-Kyoto rats. However, removing mitochondrial Ca(2+) buffering abolished these differences. A lower mitochondrial membrane potential was also observed in young SHR sympathetic neurons. This resulted in impaired mitochondrial Ca(2+) uptake and release, which might partly be responsible for the increased [Ca(2+)](i) transient and faster decay in SHR sympathetic neurons. This Ca(2+) phenotype seen in early development in cardiac stellate and superior cervical ganglion neurons may contribute to the sympathetic hyperresponsiveness that precedes the onset of hypertension.     

Alertness and psychomotor performance effects of the histamine-3 inverse agonist MK-0249 in obstructive sleep apnea patients on continuous positive airway pressure therapy with excessive daytime sleepiness: a randomized adaptive crossover study

Objectives

We aimed to evaluate the efficacy of the selective H3 receptor inverse agonist MK-0249 to treat excessive daytime sleepiness (EDS).

Methods

In this three-period, double-blind, crossover study, 125 patients (100 men, 25 women; mean age, 48.6 years) with obstructive sleep apnea receiving nasal continuous positive airway pressure therapy who had refractory EDS were randomized to 2 weeks each of daily MK-0249 (5, 8, 10, or 12 mg, adaptively assigned), modafinil 200 mg, and placebo. At baseline and after each treatment period, six maintenance of wakefulness tests (MWT) and Psychomotor Vigilance Tasks (PVT) were conducted at 2-h intervals, beginning 1 h postdose (∼09:00). The Epworth sleepiness scale (ESS), Clinical Global Impression of Severity (CGIS) and Digit Symbol Substitution Test (DSST) also were assessed. The primary end point was MWT sleep latency averaged over the first four time points (MWT-early).

Results

MWT-early mean change from baseline sleep latency at week 2 was 1.2 min for placebo, 2.1 min for MK-0249 (top two doses pooled; P > .05 vs placebo), and 5.9 min for modafinil (P ? .001 vs placebo). MK-0249 showed improvements vs placebo on secondary and exploratory end points of ESS, CGIS, PVT, and DSST. Insomnia adverse events (AEs) were greater for MK-0249 (combined doses, 17.5%) than for placebo (0.9%) or modafinil (1.8%).

Conclusion

MK-0249 did not significantly affect MWT sleep latency. However, the pattern of improvement on subjective ratings and psychomotor performance end points suggested that MK-0249 was associated with changes in aspects of cognition and performance not captured by the MWT.