Early changes in phosphatidylcholine metabolism in human acute promyelocytic leukemia cells stimulated to differentiate by phorbol ester

The HL-60 leukemia cell line derived from a human acute promyelocytic leukemia is stimulated to differentiate into macrophages within 24-28 hr after exposure to the phorbol ester, 12-O-tetradecanoylphorbol-13- acetate (TPA). We studied early alterations (within 90 min of exposure to TPA) in phosphatidylcholine metabolism in HL-60 cells and found that phosphatidylcholine synthesis by methylation is phosphatidylethanolamine was inhibited in a dose-dependent fashion. In contrast, synthesis of phosphatidylcholine from endogenous choline was enhanced and correlated inversely with the degree of inhibition of the methylation pathway. Phorbol ester congeners of TPA caused similar alterations in phosphatidylcholine metabolism in direct relationship to their capacity to induce differentiation in HL-60 cells. Perturbation of phosphatidylcholine metabolism is an early membrane even in TPA- induced HL-60 cell differentiation.     

The arterio-venous difference for immunoreactive parathyroid hormone and the production of adenosine 3,'5'-monophosphate by isolated perfused bone: studies with analogs of parathyroid hormone

Recent studies suggest that the uptake of immunoreactive parathyroid hormone (iPTH) displays different characteristics in liver, kidney, and bone. Using the isolated perfused canine bone, we have characterized the uptake of two synthetic analogs of PTH, bovine PTH-(3-34) [bPTH-3(3-34)] and [Nle8,Nle18,Tyr34]bPTH-(3-34) amide, which had previously been shown to inhibit PTH-stimulated adenylate cyclase activity in renal membranes. During the infusion of synthetic bPTH-(1-34) (3 ng/ml), extraction of iPTH by isolated perfused bone averaged 37 +/- 1%, and cAMP production rose from 6.2 +/- 2.0 to 21 +/- 3 pmol/min. Extraction of bPTH-(3-34) was similar (35 +/- 2%), but cAMP levels did not increase over baseline with PTH concentrations as high as 100 ng/ml. Simultaneous infusion of bPTH-(1-34) and bPTH-(3-34) at molar ratios of 1:2 led to a 50% inhibition of PTH-stimulated cAMP increases. The extraction by bone of the more potent in vitro inhibitor of renal cortical adenylate cyclase [Nle8,Nle18,Tyr34]bPTH-(3-34) NH2 (3 ng/ml) averaged 39 +/- 2%. In contrast, cAMP production rose from a baseline of 5.6 +/- 0.5 to 12.5 +/- 2.0 pmol/min, demonstrating agonist activity for the analog. These studies show that [Nle3,Nle18,Tyr34]bPTH-(3-34) NH2 has agonist properties in isolated perfused bone, and unsubstituted bPTh-(3-34) inhibits PTH-stimulated cAMP release by perfused bone.     

Exploring the utility of whole‐exome sequencing as a diagnostic tool in a child with atypical episodic muscle weakness

The advent of whole‐exome next‐generation sequencing (WES) has been pivotal for the molecular characterization of Mendelian disease; however, the clinical applicability of WES has remained relatively unexplored. We describe our exploration of WES as a diagnostic tool in a 3½‐year old female patient with a 2‐year history of episodic muscle weakness and paroxysmal dystonia who presented following a previous extensive but unrevealing diagnostic work‐up. WES was performed on the proband and her two parents. Parental exome data was used to filter potential de novo genomic events in the proband and suspected variants were confirmed using di‐deoxy sequencing. WES revealed a de novo non‐synonymous mutation in exon 21 of the calcium channel gene CACNA1S that has been previously reported in a single patient as a rare cause of atypical hypokalemic periodic paralysis. This was unexpected, as the proband's original differential diagnosis had included hypokalemic periodic paralysis, but clinical and laboratory features were equivocal, and standard clinical molecular testing for hypokalemic periodic paralysis and related disorders was negative. This report highlights the potential diagnostic utility of WES in clinical practice, with implications for the approach to similar diagnostic dilemmas in the future.     

Immunological response to highly active antiretroviral therapy in children with clinically stable HIV-1 infection

We studied changes in 60 immunological parameters after the administration of highly active antiretroviral therapy (HAART) in 192 clinically stable antiretroviral drug-experienced HIV-1-infected children 4 months-17 years old. The studied immunological parameters included standard lymphocyte subsets and lymphocyte surface markers of maturation and activation. The most significant changes during the 48-week study period were seen for CD8(+), CD8(+)CD62L(+)CD45RA(+), CD8(+)CD38(+)HLA-DR(+), and CD4(+) T cell percentages (P < .0001 for all parameters). These changes suggest that significant decreases in the expression of activation markers and increases in the expression of naive markers in the CD8(+) T cell population may be related to better virologic control in these HIV-1-infected children, who had relatively stable immune function at the initiation of HAART. At week 44 of HAART, the major immunological parameters in these HIV-1-infected children moved from baseline values to about halfway to two-thirds of the way toward the values in healthy, uninfected children.     

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.