Endothelin-1- and isoproterenol-induced differential protein expression and signaling pathway in HL-1 cardiomyocytes

It is well known that the two chemical compounds endothelin-1 (ET-1) and isoproterenol (ISO) can individually induce cardiac hypertrophy through G protein-coupled receptors in cardiomyocytes. However, the cardiac hypertrophy signaling pathway activated by ET-1 and ISO is not well defined. Therefore, we investigated the protein expression profile and signaling transduction in HL-l cardiomyocyte cells treated with ET-1 and ISO. Following separation of the cell lysates by using 2-DE and silver staining, we identified 16 protein spots that were differentially expressed as compared to the controls. Of these 16 spots, three changed only after treatment with ET-1, whereas four changed only after treatment with ISO, suggesting that these two stimuli could induce different signaling pathways. In order to reveal the differences between ET-1- and ISO-induced signaling, we studied the different events that occur at each step of the signaling pathways, when selected biocomponents were blocked by inhibitors. Our results indicated that ET-1 and ISO used different pathways for phosphorylation of glycogen synthase kinase-3β (GSK3β). ET-1 mainly used the mitogen-activated protein kinase and phosphatidylinositol-3-kinase/AKT pathways to activate GSK3β, whereas under ISO stimulation, only the phosphatidylinositol-3-kinase/AKT pathway was required to trigger the GSK3β pathway. Furthermore, the strength of the GSK3β signal in ISO-induced cardiac hypertrophy was stronger than that in ET-1-induced cardiac hypertrophy. We found that these two agonists brought about different changes in the protein expression of HL-1 cardiomyocytes through distinct signaling pathways even though the destination of the two signaling pathways was the same.     

Hepatic uptake and metabolism of phosphatidylcholine associated with high density lipoproteins

Background

Phosphatidylcholine (PC) is the predominant phospholipid associated with high density lipoproteins (HDL). Although the hepatic uptake of cholesteryl esters from HDL is well characterized, much less is known about the fate of PC associated with HDL. Thus, we investigated the uptake and subsequent metabolism of HDL-PC in primary mouse hepatocytes.

Methods and results

The absence of scavenger receptor-BI resulted in a 30% decrease in cellular incorporation of [³H]PC whereas [³H]cholesteryl ether uptake was almost completely abolished. Although endocytosis is not involved in the uptake of cholesteryl esters from HDL, we demonstrate that HDL internalization accounts for 40% of HDL-PC uptake. Extracellular remodeling of HDL by secretory phospholipase A₂ significantly enhances HDL lipid uptake. HDL-PC taken up by hepatocytes is partially converted to triacylglycerols via PC-phospholipase C-mediated hydrolysis of PC and incorporation of diacylglycerol into triacylglcyerol. The formation of triacylglcerol is independent of scavenger receptor-BI and occurs in extralysosomal compartments.

Conclusions and general significance

These findings indicate that HDL-associated PC is incorporated into primary hepatocytes via a pathway that differs significantly from that of HDL-cholesteryl ester, and shows that HDL-PC is more than a framework molecule, as evidenced by its partial conversion to hepatic triacylglycerol.     

The effect of hepatic lipase on coronary artery disease in humans is influenced by the underlying lipoprotein phenotype

Increased or decreased hepatic lipase (HL) activity has been associated with coronary artery disease (CAD). This is consistent with the findings that gene variants that influence HL activity were associated with increased CAD risk in some population studies but not in others. In this review, we will explain the conditions that influence the effects of HL on CAD. Increased HL is associated with smaller and denser LDL (sdLDL) and HDL (HDL₃) particles, while decreased HL is associated with larger and more buoyant LDL and HDL particles. The effect of HL activity on CAD risk is dependent on the underlying lipoprotein phenotype or disorder. Central obesity with hypertriglyceridemia (HTG) is associated with high HL activity that leads to the formation of sdLDL that is pro-atherogenic. In the absence of HTG, where large buoyant cholesteryl ester-enriched LDL is prominent, elevation of HL does not raise the risk for CAD. In HTG patients, drug therapy that decreases HL activity selectively decreases sdLDL particles, an anti-atherogenic effect. Drug therapy that raises HDL₂ cholesterol has not decreased the risk for CAD. In trials where inhibition of cholesterol ester transfer protein (CETP) or HL occurs, the increase in HDL₂ most likely is due to inhibition of catabolism of HDL₂ and impairment of reverse cholesterol transport (RCT). In patients with isolated hypercholesterolemia, but with normal triglyceride levels and big-buoyant LDL particles, an increase in HL activity is beneficial; possibly because it increases RCT. Drugs that lower HL activity might decrease the risk for CAD only in hypertriglyceridemic patients with sdLDL by selectively clearing sdLDL particles from plasma, which would override the potentially pro-atherogenic effect on RCT. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Genome wide analysis in a family with sensorineural hearing loss,autism and mental retardation

Hearing loss is a common congenital anomaly with an incidence of 1 in 1000 live births. It has been described together with several other clinical features as fortuitous association or commune genetic syndrome. In this study, we investigated a consanguineous Tunisian family with moderate to profound congenital hearing loss, mental retardation and autistic behaviors. We performed a genome wide microarray analysis study using approximately 300,000 SNPs in a common set of 7 invidious of this family. We identified regions of suggestive linkage with hearing loss on chromosomes 6p12 and 7q34. In addition, we identified a deletion on chromosome 8p in the two autistic individuals. This report presents an illustration of how consanguinity could increase familial clustering of multiple hereditary diseases within the same family. The application of next generation sequencing for this family seems to be a good strategy for further analysis leading to the identification of candidate genes.     

Robust thermoregulatory overcompensation, rather than tolerance, develops with serial administrations of 70% nitrous oxide to rats

Changes in typical whole-animal dependent variables following drug administration represent an integral of the drug's pharmacological effect, the individual's autonomic and behavioral responses to the resulting disturbance, and many other influences. An archetypical example is core temperature (T_c), long used for quantifying initial drug sensitivity and tolerance acquisition over repeated drug administrations. Our previous work suggested that rats differing in initial sensitivity to nitrous oxide (N₂O)-induced hypothermia would exhibit different patterns of tolerance development across N₂O administrations. Specifically, we hypothesized that rats with an initially insensitive phenotype would subsequently develop regulatory overcompensation that would mediate an allostatic hyperthermic state, whereas rats with an initially sensitive phenotype would subsequently compensate to a homeostatic normothermic state. To preclude confounding due to handling and invasive procedures, a valid test of this prediction required non-invasive thermal measurements via implanted telemetric temperature sensors, combined direct and indirect calorimetry, and automated drug delivery to enable repeatable steady-state dosing. We screened 237 adult rats for initial sensitivity to 70% N₂O-induced hypothermia. Thirty highly sensitive rats that exhibited marked hypothermia when screened and 30 highly insensitive rats that initially exhibited minimal hypothermia were randomized to three groups (n=10 each/group) that received: (1) twelve 90-min exposures to 70% N₂O using a classical conditioning procedure, (2) twelve 90-min exposures to 70% N₂O using a random control procedure for conditioning, or (3) a no-drug control group that received custom-made air. Metabolic heat production (via indirect calorimetry), body heat loss (via direct calorimetry) and T_c (via telemetry) were simultaneously quantified during N₂O and control gas administrations. Initially insensitive rats rapidly acquired (3rd administration) a significant allostatic hyperthermic phenotype during N₂O administration whereas initially sensitive rats exhibited classical tolerance (normothermia) during N₂O inhalation in the 4th and 5th sessions. However, the sensitive rats subsequently acquired the hyperthermic phenotype and became indistinguishable from initially insensitive rats during the 11th and 12th N₂O administrations. The major mechanism for hyperthermia was a brisk increase in metabolic heat production. However, we obtained no evidence for classical conditioning of thermal responses. We conclude that the degree of initial sensitivity to N₂O-induced hypothermia predicts the temporal pattern of thermal adaptation over repeated N₂O administrations, but that initially insensitive and sensitive animals eventually converge to similar (and substantial) magnitudes of within-administration hyperthermia mediated by hyper-compensatory heat production.     

Luminol‐dependent chemiluminescence of human phagocyte cell lines: comparison between DMSO differentiated PLB 985 and HL 60 cells

The human promyelocytic leukemia HL 60 and PLB 985 cell lines can differentiate into terminally mature neutrophil‐like cells via dimethyl sulfoxide (DMSO) induction. In this study the luminol‐dependent chemiluminescence (LCL) of both neutrophil‐like cells was analayzed and compared in response to phorbol myristate acetate (PMA) and opsonized zymosan (OZ) stimulants. It was shown that, like human blood neutrophils, both neutrophil‐like cells expressed high levels of CD11b, but unlike human blood neutrophils these cells almost lack LCL‐detectable intracellular oxidase activity. By studying the pattern of activation to OZ and PMA and priming with GM‐CSF, we concluded that there is no difference between the percentage of differentiation and function of DMSO‐induced HL 60 and PLB 985. However, the LCL capacity (area under the curve) of DMSO induced PLB 985 cells was higher than that of HL 60 cells in response to both PMA and OZ, which implies a higher capacity to generate reactive oxygen species in PLB 985 cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Retinoic acid treated HL60 cells express CEACAM1 (CD66a) and phagocytose Neisseria gonorrhoeae

Neisseria gonorrhoeae (gonococci, GC) are phagocytosed by neutrophils through the interaction between opacity proteins (Opa) and the CEA (CD66) family of antigens. In order to study this interaction, we used the human myeloid leukemia HL60 cell line, which differentiates into granulocyte-like cells upon treatment with dimethylsulfoxide (DMSO) or retinoic acid (RA). We found that RA-, but not DMSO- or untreated-HL60 cells, can phagocytose OpaI-expressing gonococci as well as Escherichia coli. The interaction of OpaI E. coli with RA-treated HL60 cells was inhibited by antibodies against CEACAM1. Phagocytosis of OpaI E. coli was found to be a result of the expression of CEACAM1 in RA-treated HL60 cells. Our results indicate that the level of expression of CEACAM1 in HL60 cells can be regulated by treatment with RA in a differentiation-dependent manner, and that this is important for phagocytosis of OpaI-expressing gonococci or E. coli.     

The occurrence of rapidly reversible non-photochemical quenching of chlorophyll a fluorescence in cyanobacteria

Cyanobacteria have previously been considered to differ fundamentally from plants and algae in their regulation of light harvesting. We show here that in fact the ecologically important marine prochlorophyte, Prochlorococcus, is capable of forming rapidly reversible non-photochemical quenching of chlorophyll a fluorescence (NPQf or qE) as are freshwater cyanobacteria when they employ the iron stress induced chlorophyll-based antenna, IsiA. For Prochlorococcus, the capacity for NPQf is greater in high light-adapted strains, except during iron starvation which allows for increased quenching in low light-adapted strains. NPQf formation in freshwater cyanobacteria is accompanied by deep Fo quenching which increases with prolonged iron starvation.     

Carboxy-terminal fragment of osteogenic growth peptide regulates myeloid differentiation through RhoA

The carboxy-terminal fragment of osteogenic growth peptide, OGP(10-14), is a pentapeptide with bone anabolic effects and hematopoietic activity. The latter activity appears to be largely enhanced by specific growth factors. To study the direct activity of OGP(10-14) on myeloid cells, we tested the pentapeptide proliferating/differentiating effects in HL60 cell line. In this cell line, OGP(10-14) significantly inhibited cell proliferation, and enhanced myeloperoxidase (MPO) activity and nitroblue tetrazolium reducing ability. Moreover, it induced cytoskeleton remodeling and small GTP-binding protein RhoA activation. RhoA, which is known to be involved in HL60 differentiation, mediated these effects as shown by using its specific inhibitor, C3. Treatment with GM-CSF had a comparable OGP(10-14) activity on proliferation, MPO expression, and RhoA activation. Further studies on cell proliferation and RhoA activation proved enhanced activity by association of the two factors. These results strongly suggest that OGP(10-14) acts directly on HL60 cells by activating RhoA signaling although other possibilities cannot be ruled out.     

Apoptosis induced kinetic changes in autofluorescence of cultured HL60 cells-possible application for single cell analysis on chip

INTRODUCTION: This paper presents a new method using natural cellular fluorescence (autofluorescence, AF) to study apoptosis. Measurement of AF reduces sample preparation time and avoids cellular toxicity due to the fact that no labelling is required. METHODS: Human promyelocytic leukemic HL60 cells were incubated with camptothecin (CPT), tumour necrosis factor (TNF)-alpha in combination with cycloheximide (CHX), or irradiated with 6 or 10 Gray, during varying time periods, to initiate apoptosis. AF was measured at the flow cytometer. RESULTS: Induction of apoptosis results in the shrinkage of the cell and the fragmentation into apoptotic bodies. With flow cytometry, 4 subpopulations, viable, early apoptotic, late apoptotic and the necrotic cells, can be distinguished. Induction of apoptosis results in a decrease in AF intensity compared to untreated HL60 cells, especially seen in the late apoptotic subpopulation. The AF intensity is found to decrease significantly in time (between 2 h and 24 h) for all the four apoptotic inducers used. CONCLUSIONS: Our results show that it is possible to specifically measure the apoptotic-induced kinetic changes in AF in HL60 cells. A decrease in AF intensity is seen from 2 h till 24 h. These results open a door for future developments in single-cell analysis.