Inhibition of proliferation and induction of apoptosis in juvenile myelomonocytic leukemic cells by the granulocyte-macrophage colony-stimulating factor analogue E21R

Juvenile myelomonocytic leukemia (JMML) is a malignancy that almost inevitably leads to death before adulthood. Chemotherapy has given disappointing results and a substantial number of patients relapse after bone marrow transplantation. A salient feature of this disease is that the JMML cells produce granulocyte-macrophage colony-stimulating factor (GM-CSF) spontaneously and survive and proliferate without exogeneous GM-CSF. Furthermore, JMML cells are hypersensitive to GM-CSF with addition of this cytokine leading to enhanced proliferation. We have recently generated a human GM-CSF analogue, E21R, that acts as a complete and selective GM-CSF receptor antagonist. We have now tested this molecule as a potential new agent to control the leukemic cell load in JMML with particular emphasis on its role in JMML cell survival. We found that E21R inhibited the spontaneous growth of JMML cells in vitro and caused their apoptosis in a dose- and time-dependent manner in seven of seven cases. In contrast, neither a neutralizing anti-GM-CSF monoclonal antibody (MoAb) nor a selective interleukin-1 (IL-1) receptor antagonist affected JMML cell survival. Furthermore, the apoptotic effect of E21R was seen even in the presence of interleukin-1 beta and tumor necrosis factor-alpha, which have also been implicated in the pathogenesis of JMML. The inhibitory effects of E21R on JMML cell growth and viability offer a novel approach to therapy in this lethal childhood leukemia.     

Inositol hexakisphosphate stimulates non-Ca2+-mediated and primes Ca2+-mediated exocytosis of insulin by activation of protein kinase C

D-myo-inositol 1,2,3,4,5,6-hexakisphosphate (InsP6), formed via complex pathways of inositol phosphate metabolism, composes the main bulk of inositol polyphosphates in the cell. Relatively little is known regarding possible biological functions for InsP6. We now show that InsP6 can modulate insulin exocytosis in permeabilized insulin-secreting cells. Concentrations of InsP6 above 20 microM stimulated insulin secretion at basal Ca2+-concentration (30 nM) and primed Ca2+-induced exocytosis (10 microM), both effects being due to activation of protein kinase C. Our results suggest that InsP6 can play an important modulatory role in the regulation of processes such as exocytosis in insulin-secreting cells. The specific role for InsP6 can then be to recruit secretory granules to the site of exocytosis.     

Isolation and characterization of rat liver amphisomes. Evidence for fusion of autophagosomes with both early and late endosomes

Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between autophagosomes and endosomes, have so far only been characterized in indirect, functional terms. To enable a physical distinction between autophagosomes and amphisomes, the latter were selectively density-shifted in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prior to subcellular fractionation). Whereas amphisomes, by this criterion, accounted for only a minor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the AV population. A quantitative electron microscopic study confirmed that leupeptin induced a severalfold increase in the number of hepatocytic amphisomes (recognized by their gold particle contents; otherwise, their ultrastructure was quite similar to autophagosomes). Leupeptin caused, furthermore, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amphisome-lysosome fusion. The electron micrographs suggested that autophagosomes could undergo multiple independent fusions, with multivesicular (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphisomes were enriched in early endosome markers (the asialoglycoprotein receptor and the early endosome-associated protein 1) as well as in a late endosome marker (the cation-independent mannose 6-phosphate receptor). Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes.     

Transarterial embolization of the uterine arteries: patient reactions and effects on uterine vasculature

Corticotropin releasing hormone (CRH) has been localized to interneurons of the mammalian cerebral cortex, but these neurons have not been fully characterized. The present study determined the extent of co-localization of CRH with glutamate decarboxylase (GAD) and calcium-binding proteins in the infant rat neocortex using immunocytochemistry. CRH-immunoreactive (ir) neurons were classified into two major groups. The first group was larger and consisted of densely CRH-immunostained small bipolar cells, predominantly localized to layers II and III. The second group of CRH-ir cells was lightly labeled and included multipolar neurons mainly found in deep cortical layers. Co-localization studies indicated that the vast majority of CRH-ir neurons, including both bipolar and multipolar types, was co-immunolabeled for GAD-65 and GAD-67. Most multipolar, but only some bipolar, CRH-ir neurons also contained parvalbumin, while CRH-ir neurons rarely contained calbindin or calretinin. These results indicate that virtually all CRH-ir neurons in the rat cerebral cortex are GABAergic. Furthermore, since parvalbumin is expressed by cortical basket and chandelier cells, the co-localization of CRH and parvalbumin suggests that some cortical CRH-ir neurons may belong to these two cell types.     

Expression of p53 in reactive mesothelium

An arteriovenous fistula originating from halfway down the circumflex coronary artery, draining into the coronary sinus was first diagnosed in a patient of 81. For 10 years, she had had dyspnea on exertion, mild mitral regurgitation and permanent ventricular pacing for symptomatic bradyarrhythmia. She was treated medically and despite her age was offered transcatheter balloon embolization, but she refused to undergo such intervention.     

Kinetic properties and stereospecificity of the monomeric dUTPase from herpes simplex virus type 1

The heptadecapeptide orphanin FQ or nociceptin (OFQ/N), the endogenous ligand for the orphan opioid receptor, has a complex pharmacology in mice, eliciting either an anti-opioid/hyperalgesic action or analgesia depending upon the dose and testing paradigm. Unlike mice, orphanin FQ/nociceptin fails to elicit hyperalgesia in the rat following intracerebroventricular injection. Both OFQ/N and a truncated version, OFQ/N(1-11), produce a robust analgesic response. OFQ/N analgesia is readily antagonized by the opioid antagonists naloxone or diprenorphine, despite their very poor affinity for the cloned orphan opioid receptor. Antisense studies revealed that probes targeting the second and third coding exon of the orphan clone significantly attenuate OFQ/N analgesia, while the exon 1 probe was inactive. These results indicate that OFQ/N elicits a naloxone-sensitive analgesia in rats similar to that previously reported in mice.     

Nutrient intake and blood pressure in the Dietary Intervention Study in Children

Delineating the role that diet plays in blood pressure levels in children is important for guiding dietary recommendations for the prevention of hypertension. The purpose of this study was to investigate relationships between dietary nutrients and blood pressure in children. Data were analyzed from 662 participants in the Dietary Intervention Study in Children who had elevated low-density lipoprotein cholesterol and were aged 8 to 11 years at baseline. Three 24-hour dietary recalls, systolic pressure, diastolic pressure, height, and weight were obtained at baseline, 1 year, and 3 years. Nutrients analyzed were the micronutrients calcium, magnesium, and potassium; the macronutrients protein, carbohydrates, total fat, saturated fat, polyunsaturated fat, and monounsaturated fat; dietary cholesterol; and total dietary fiber. Baseline and 3-year longitudinal relationships were examined through multivariate models on diastolic and systolic pressures separately, controlling for height, weight, sex, and total caloric intake. The following associations were found in longitudinal analyses: analyzing each nutrient separately, for systolic pressure, inverse associations with calcium (P < .05); magnesium, potassium, and protein (all P < .01); and fiber (P < .05), and direct associations with total fat and monounsaturated fat (both P < .05); for diastolic pressure, inverse associations with calcium (P < .01); magnesium and potassium (both P < .05), protein (P < .01); and carbohydrates and fiber (both P < .05), and direct associations with polyunsaturated fat (P < .01) and monounsaturated fat (P < .05). Analyzing all nutrients simultaneously, for systolic pressure, direct association with total fat (P < .01); for diastolic pressure, inverse associations with calcium (P < .01) and fiber (P < .05), and direct association with total and monounsaturated fats (both P < .05). Results from this sample of children with elevated low-density lipoprotein cholesterol indicate that dietary calcium, fiber, and fat may be important determinants of blood pressure level in children.     

Insulin-like growth factor II signaling through the insulin-like growth factor II/mannose-6-phosphate receptor promotes exocytosis in insulin-secreting cells

The insulin-like growth factor II (IGF-II)/mannose-6-phosphate (M-6-P) receptor is known to participate in endocytosis as well as sorting of lysosomal enzymes and is involved in membrane trafficking through rapid cycling between cytosolic membrane compartments and the plasma membrane. Here we demonstrate that IGF-II, acting through the IGF-II/M-6-P receptor, promotes exocytosis of insulin in the pancreatic beta cell. The effect of IGF-II was evoked at nonstimulatory concentrations of glucose, was mediated by a pertussis toxin sensitive GTP-binding protein, was dependent on protein kinase C-induced phosphorylation, and was independent of changes in cytoplasmic free Ca2+ concentration. Since the applied concentration of IGF-II is within the range normally found free in circulation in humans, this novel signaling pathway for the IGF-II/M-6-P receptor is likely to be involved in modulation of insulin exocytosis under physiological conditions.