Clinical characteristics and plasma lipids in subjects with familial combined hypolipidemia: a pooled analysis

Angiopoietin-like 3 (ANGPTL3) regulates lipoprotein metabolism by modulating extracellular lipases. Loss-of function mutations in ANGPTL3 gene cause familial combined hypolipidemia (FHBL2). The mode of inheritance and hepatic and vascular consequences of FHBL2 have not been fully elucidated. To get further insights on these aspects, we reevaluated the clinical and the biochemical characteristics of all reported cases of FHBL2. One hundred fifteen FHBL2 individuals carrying 13 different mutations in the ANGPTL3 gene (14 homozygotes, 8 compound heterozygotes, and 93 heterozygotes) and 402 controls were considered. Carriers of two mutant alleles had undetectable plasma levels of ANGPTL3 protein, whereas heterozygotes showed a reduction ranging from 34% to 88%, according to genotype. Compared with controls, homozygotes as well as heterozygotes showed a significant reduction of all plasma lipoproteins, while no difference in lipoprotein(a) [Lp(a)] levels was detected between groups. The prevalence of fatty liver was not different in FHBL2 subjects compared with controls. Notably, diabetes mellitus and cardiovascular disease were absent among homozygotes. FHBL2 trait is inherited in a codominant manner, and the lipid-lowering effect of two ANGPTL3 mutant alleles was more than four times larger than that of one mutant allele. No changes in Lp(a) were detected in FHBL2. Furthermore, our analysis confirmed that FHBL2 is not associated with adverse clinical sequelae. The possibility that FHBL2 confers lower risk of diabetes and cardiovascular disease warrants more detailed investigation.     

Multiple presentation of Scfv800E6 on silica nanospheres enhances targeting efficiency toward HER-2 receptor in breast cancer cells

Spherical silica nanoparticles (SNP) have been synthesized and functionalized with anti-HER-2 scFv800E6 antibody by both localized histidine-tag recognition, leading to an oriented protein ligation, and glutaraldehyde cross-linking, exploiting a statistical reactivity of lysine amine groups in the primary sequence of the molecule. The targeting efficiency of nanocomplexes in comparison with free scFv was evaluated by flow cytometry using a HER-2 antigen-positive MCF-7 breast cancer cell line, exhibiting a 4-fold increase in scFv binding efficacy, close to the affinity of intact anti-HER-2 monoclonal antibody, which suggests the effectiveness of presenting multiple scFv molecules on nanoparticles in improving antigen recognition. Unexpectedly, the conjugation method did not affect the binding efficacy of scFv, suggesting a structural role of lysines in the scFv molecule. Confocal laser scanning microscopy confirmed the binding of nanocomplexes to HER-2 and also provided evidence of their localization at the cell surface.     

Synthesis of 7-oxo-7H-naphtho[1,2,3-de]quinoline derivatives as potential anticancer agents active on multidrug resistant cell lines

Following our earlier finding that tetracyclic anthraquinone analogs with a fused pyridone ring exhibit cytotoxic activity toward multidrug resistant tumor cells, a series of new potential antitumor agents, 7-oxo-7H-naphtho[1,2,3-de]quinoline derivatives (3, 6-8, 10-12, 14, 15, and 18), bearing one or two basic side chains and various substituents at the pyridone ring, have been synthesized. The compounds have been obtained from 1-amino-4-chloroanthraquinone or 1-aminoanthraquinone by cyclization with diethyl malonate and the subsequent reactions of the key intermediates 2, 4, and 17. The compounds exhibited cytotoxic activity toward sensitive human leukemia cell line HL-60 and against its resistant sublines HL-60/VINC (MDR1 type) and HL-60/DX (MRP1 type).     

AQP4-Dependent Water Transport Plays a Functional Role in Exercise-Induced Skeletal Muscle Adaptations

In this study we assess the functional role of Aquaporin-4 (AQP4) in the skeletal muscle by analyzing whether physical activity modulates AQP4 expression and whether the absence of AQP4 has an effect on osmotic behavior, muscle contractile properties, and physical activity. To this purpose, rats and mice were trained on the treadmill for 10 (D10) and 30 (D30) days and tested with exercise to exhaustion, and muscles were used for immunoblotting, RT-PCR, and fiber-type distribution analysis. Taking advantage of the AQP4 KO murine model, functional analysis of AQP4 was performed on dissected muscle fibers and sarcolemma vesicles. Moreover, WT and AQP4 KO mice were subjected to both voluntary and forced activity. Rat fast-twitch muscles showed a twofold increase in AQP4 protein in D10 and D30 rats compared to sedentary rats. Such increase positively correlated with the animal performance, since highest level of AQP4 protein was found in high runner rats. Interestingly, no shift in muscle fiber composition nor an increase in AQP4-positive fibers was found. Furthermore, no changes in AQP4 mRNA after exercise were detected, suggesting that post-translational events are likely to be responsible for AQP4 modulation. Experiments performed on AQP4 KO mice revealed a strong impairment in osmotic responses as well as in forced and voluntary activities compared to WT mice, even though force development amplitude and contractile properties were unvaried. Our findings definitively demonstrate the physiological role of AQP4 in supporting muscle contractile activity and metabolic changes that occur in fast-twitch skeletal muscle during prolonged exercise.     

Detailing Radio Frequency Heating Induced by Coronary Stents: A 7.0 Tesla Magnetic Resonance Study

The sensitivity gain of ultrahigh field Magnetic Resonance (UHF-MR) holds the promise to enhance spatial and temporal resolution. Such improvements could be beneficial for cardiovascular MR. However, intracoronary stents used for treatment of coronary artery disease are currently considered to be contra-indications for UHF-MR. The antenna effect induced by a stent together with RF wavelength shortening could increase local radiofrequency (RF) power deposition at 7.0 T and bears the potential to induce local heating, which might cause tissue damage. Realizing these constraints, this work examines RF heating effects of stents using electro-magnetic field (EMF) simulations and phantoms with properties that mimic myocardium. For this purpose, RF power deposition that exceeds the clinical limits was induced by a dedicated birdcage coil. Fiber optic probes and MR thermometry were applied for temperature monitoring using agarose phantoms containing copper tubes or coronary stents. The results demonstrate an agreement between RF heating induced temperature changes derived from EMF simulations versus MR thermometry. The birdcage coil tailored for RF heating was capable of irradiating power exceeding the specific-absorption rate (SAR) limits defined by the IEC guidelines by a factor of three. This setup afforded RF induced temperature changes up to +27 K in a reference phantom. The maximum extra temperature increase, induced by a copper tube or a coronary stent was less than 3 K. The coronary stents examined showed an RF heating behavior similar to a copper tube. Our results suggest that, if IEC guidelines for local/global SAR are followed, the extra RF heating induced in myocardial tissue by stents may not be significant versus the baseline heating induced by the energy deposited by a tailored cardiac transmit RF coil at 7.0 T, and may be smaller if not insignificant than the extra RF heating observed under the circumstances used in this study.     

Synthesis and antiproliferative activity of alkylphosphocholines

Alkylphosphocholines (APC) with one or more methylene groups in the alkyl chain replaced by oxygen atoms or carbonyl groups, or both have been assembled modularly using omega-diols as central building blocks. Out of 25 new compounds of this kind, 11 were evaluated for their antiproliferative activity on four cell lines and compared with miltefosine to evaluate their hemolytic activity (HA) and cytotoxicity on non-tumoral cells (MT2), used as markers of adverse effects. Compound 13 was more active on cancer cell lines than on non-tumoral cells and the data were similar for MTT and thymidine incorporation assays. It had less HA than miltefosine. Compound 13 could therefore be a candidate for the preparation of compounds with higher cytotoxicity on cancer cells and lower general toxicity.     

Up-regulation of nuclear PLCbeta1 in myogenic differentiation

Phospholipase C beta(1) (PLCbeta(1)) signaling in both cell proliferation and differentiation has been largely investigated, but its role in myoblast differentiation is still unclear. The C2C12 myogenic cell line has been used in this study in order to find out the role of the two subtypes of PLCbeta(1), i.e., a and b in this process. C2C12 myoblast proliferate in response to mitogens and upon mitogen withdrawal differentiates into multinucleated myotubes. We found that differentiation of C2C12 skeletal muscle cells is characterized by a marked increase in the amount of nuclear PLCbeta(1)a and PLCbeta(1)b. Indeed, treatment with insulin induces a dramatic rise of both PLCbeta(1) subtypes expression and activity, as determined by immunochemical and enzymatic assays. Immunofluorescence experiments with anti-PLCbeta(1) specific monoclonal antibody showed a low level of cytoplasmatic and nuclear staining during the initial 12 h of differentiation whilst a massive nuclear staining is appreciable in differentiating cells. The time course of PLCbeta(1) expression versus Troponin T expression clearly indicates that the increase in the amount of PLCbeta(1) takes place 24 h earlier than that of Troponin T. Moreover, the overexpression of the PLCbeta(1)M2b mutant, lacking the nuclear localization signal and entirely located in the cytoplasm, represses the formation of mature multinucleated myotube. Taken together these results suggest that nuclear PLCbeta(1) is a key player in myoblast differentiation, functioning as a positive regulator of this process.     

Prediction of the disulfide-bonding state of cysteines in proteins at 88% accuracy

The task of predicting the cysteine-bonding state in proteins starting from the residue chain is addressed by implementing a new hybrid system that combines a neural network and a hidden Markov model (hidden neural network). Training is performed using 4136 cysteine-containing segments extracted from 969 nonhomologous proteins of well-resolved three-dimensional structure. After a 20-fold cross-validation procedure, the efficiency of the prediction scores as high as 88% and 84%, when measured on cysteine and protein basis, respectively. These results outperform previously described methods for the same task.     

Nuclear diacylglycerol kinase-theta is activated in response to nerve growth factor stimulation of PC12 cells

Previous evidence from independent laboratories has shown that the nucleus contains diacylglycerol kinase (DGK) isoforms, i.e., the enzymes, which yield phosphatidic acid from diacylglycerol, thus terminating protein kinase C-mediated signaling events. A DGK isoform, which resides in the nucleus of PC12 cells, is DGK-theta. Here, we show that nerve growth factor (NGF) treatment of serum-starved PC12 cells results in the stimulation of both a cytoplasmic and a nuclear DGK activity. However, time course analysis shows that cytoplasmic DGK activity peaked earlier than its nuclear counterpart. While nuclear DGK activity was dramatically down-regulated by a monoclonal antibody known for selectively inhibiting DGK-theta, cytoplasmic DGK activity was not. Moreover, nuclear DGK activity was stimulated by phosphatidylserine, an anionic phospholipid that had no effect on cytoplasmic DGK activity. Upon NGF stimulation, the amount and the activity of DGK-theta, which was bound to the insoluble nuclear matrix fraction, substantially increased. Epidermal growth factor up-regulated a nuclear DGK activity insensitive to anti-DGK-theta monoclonal antibody. Overall, our findings identify nuclear DGK-theta as a down-stream target of NGF signaling in PC12 cells.