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.     

Lipoprotein lipase isoelectric point isoforms in humans

Lipoprotein lipase (LPL) hydrolyzes circulating triacylglycerols (TAG) into free fatty acids and glycerol. It is present in almost all tissues and its tissue-specific regulation directs the flow of circulating TAG in the body. We demonstrated in a previous study that, in rat heart and post-heparin plasma (PHP), LPL consists of a pattern of more than 8 forms of the same apparent molecular weight, but different isoelectric point (pI). In the present study we describe, for the first time, the existence of at least nine LPL pI isoforms in human PHP, with apparent pI between 6.8 and 8.6. Separation and characterization of these forms was carried out by 2DE combined with Western blotting and mass spectrometry (MALDI-TOF/MS and LC–MS/MS). Further studies are needed to discover their molecular origin, the pattern of pI isoforms in human tissues, their possible physiological functions and possible modifications of their pattern in different pathologies.     

Expression patterns of QTL based and other candidate genes in Madhukar × Swarna RILs with contrasting levels of iron and zinc in unpolished rice grains

Background

Identifying QTLs/genes for iron and zinc in rice grains can help in biofortification programs. Genome wide mapping showed 14 QTLs for iron and zinc concentration in unpolished rice grains of F7 RILs derived from Madhukar × Swarna. One line (HL) with high Fe and Zn and one line (LL) with low Fe and Zn in unpolished rice were compared with each other for gene expression using qPCR. 7 day old seedlings were grown in Fe + and Fe − medium for 10 days and RNA extracted from roots and shoots to determine the response of 15 genes in Fe − conditions.

Results

HL showed higher upregulation than LL in shoots but LL showed higher upregulation than HL in roots. YSL2 was upregulated only in HL roots and YSL15 only in HL shoots and both up to 60 fold under Fe − condition. IRT2 and DMAS1 were upregulated 100 fold and NAS2 1000 fold in HL shoot. NAS2, IRT1, IRT2 and DMAS1 were upregulated 40 to 100 fold in LL roots. OsZIP8, OsNAS3, OsYSL1 and OsNRAMP1 which underlie major Fe QTL showed clear allelic differences between HL and LL for markers flanking QTL. The presence of iron increasing QTL allele in HL was clearly correlated with high expression of the underlying gene. OsZIP8 and OsNAS3 which were within major QTL with increasing effect from Madhukar were 8 fold and 4 fold more expressed in HL shoot than in LL shoot. OsNAS1, OsNAS2, OsNAS3, OsYSL2 and OsYSL15 showed 1.5 to 2.5 fold upregulation in flag leaf of HL when compared with flag leaf of Swarna.

Conclusion

HL and LL differed in root length, Fe concentration and expression of several genes under Fe deficiency. The major distinguishing genes were NAS2, IRT2, DMAS1, and YSL15 in shoot and NAS2, IRT1, IRT2, YSL2, and ZIP8 in roots. The presence of iron increasing QTL allele in HL at marker locus close to genes also increased upregulation in HL.     

Towards a critical understanding of the photosystem II repair mechanism and its regulation during stress conditions

Photosystem II (PSII) is vulnerable to high light (HL) illumination resulting in photoinhibition. In addition to photoprotection mechanisms, plants have developed an efficient PSII repair mechanism to save themselves from irreversible damage to PSII under abiotic stresses including HL illumination. The phosphorylation/dephosphorylation cycle along with subsequent degradation of photodamaged D1 protein to be replaced by the insertion of a newly synthesized copy of D1 into the PSII complex, is the core function of the PSII repair cycle. The exact mechanism of this process is still under discussion. We describe the recent progress in identifying the kinases, phosphatases and proteases, and in understanding their involvement in the maintenance of thylakoid structure and the quality control of proteins by PSII repair cycle during photoinhibition.     

Update of the spectrum of GJB2 gene mutations in Tunisian families with autosomal recessive nonsyndromic hearing loss

Hearing loss is the most frequent sensory disorder. It affects 3 in 1000 newborns. It is genetically heterogeneous with 60 causally-related genes identified to date. Mutations in GJB2 gene account for half of all cases of non-syndromic deafness. The aim of this study was to determine the relative frequency of GJB2 allele variants in Tunisia. In this study, we screened 138 patients with congenital hearing loss belonging to 131 families originating from different parts of Tunisia for mutations in GJB2 gene. GJB2 mutations were found in 39% of families (51/131). The most common mutation was c.35delG accounting for 35% of all cases (46/131). The second most frequent mutation was p.E47X present in 3.8% of families. Four identified mutations in our cohort have not been reported in Tunisia; p.V37I, c.235delC, p.G130A and the splice site mutation IVS1+1G>A (0.76%). These previously described mutations were detected only in families originating from Northern and not from other geographical regions in Tunisia. In conclusion we have confirmed the high frequency of c.35delG in Tunisia which represents 85.4% of all GJB2 mutant alleles. We have also extended the mutational spectrum of GJB2 gene in Tunisia and revealed a more pronounced allelic heterogeneity in the North compared to the rest of the country.     

Combined Fourier transform infrared and Raman spectroscopic approach for identification of multidrug resistance phenotype in cancer cell lines

Cancer cells escape cytotoxic effects of anticancer drugs by a process known as multidrug resistance (MDR). Identification of cell status by less time-consuming methods can be extremely useful in patient management and treatment. This study aims at evaluating the potentials of vibrational spectroscopic methods to perform cell typing and to differentiate between sensitive and resistant human cancer cell lines, in particular those that exhibit the MDR phenotype. Micro-Raman and Fourier transform infrared (FTIR) spectra have been acquired from the sensitive promyelocytic HL60 leukemia cell line and two of its subclones resistant to doxorubicin (HL60/DOX) and daunorubicin (HL60/DNR), and from the sensitive MCF7 breast cancer cell line and its MDR counterpart resistant to verapamil (MCF7/VP). Principal components analysis (PCA) was employed for spectral comparison and classification. Our data show that cell typing was feasible with both methods, giving two distinct clusters for HL60- and MCF7-sensitive cells. In addition, phenotyping of HL60 cells, i.e., discriminating between the sensitive and MDR phenotypes, was attempted by both methods. FTIR could not only delineate between the sensitive and resistant HL60 cells, but also gave two distinct clusters for the resistant cells, which required a two-step procedure with Raman spectra. In the case of MCF7 cell lines, both the sensitive and resistant phenotypes could be differentiated very efficiently by PCA analysis of their FTIR and Raman point spectra. These results indicate the prospective applicability of FTIR and micro-Raman approaches in the differentiation of cell types as well as characterization of the cell status, such as the MDR phenotype exhibited in resistant leukemia cell lines like HL60 and MCF7.     

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.     

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.