The Obesity Paradox: A Misleading Term That Should Be Abandoned

The term “obesity paradox” is a figure of speech, not a scientific term. The term has no precise definition and has been used to describe numerous observations that have little in common other than the finding of an association of obesity with a favorable outcome. The terminology has led to misunderstandings among researchers and the public alike. It's time for authors and editors to abandon the use of this term. Simply labeling counterintuitive findings as the “obesity paradox” adds no value. Unexpected findings should not be viewed negatively; such findings can lead to new knowledge, better treatments, and scientific advances.     

HU histone-like DNA-binding protein from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>: structural and evolutionary analyses

The histone-like DNA-binding proteins (HU) serve as model molecules for protein thermostability studies, as they function in different bacteria that grow in a wide range of temperatures and show sequence diversity under a common fold. In this work, we report the cloning of the hutth gene from Thermus thermophilus, the purification and crystallization of the recombinant HUTth protein, as well as its X-ray structure determination at 1.7 Å. Detailed structural and thermodynamic analyses were performed towards the understanding of the thermostability mechanism. The interaction of HUTth protein with plasmid DNA in solution has been determined for the first time with MST. Sequence conservation of an exclusively thermophilic order like Thermales, when compared to a predominantly mesophilic order (Deinococcales), should be subject, to some extent, to thermostability-related evolutionary pressure. This hypothesis was used to guide our bioinformatics and evolutionary studies. We discuss the impact of thermostability adaptation on the structure of HU proteins, based on the detailed evolutionary analysis of the Deinococcus–Thermus phylum, where HUTth belongs. Furthermore, we propose a novel method of engineering thermostable proteins, by combining consensus-based design with ancestral sequence reconstruction. Finally, through the structure of HUTth, we are able to examine the validity of these predictions. Our approach represents a significant advancement, as it explores for the first time the potential of ancestral sequence reconstruction in the divergence between a thermophilic and a mainly mesophilic taxon, combined with consensus-based engineering.     

Matrix metalloproteinase inhibitors as anticancer agents

The important role of matrix metalloproteinases (MMPs) in the process of carcinogenesis is well established. However, despite very promising activity in a plethora of preclinical models, MMP inhibitors (MMPIs) failed to demonstrate a statistically significant survival advantage in advanced stage clinical trials in most human malignancies. Herein, we review the implication of MMPs in carcinogenesis, outline the pharmacology and current status of various MMPIs as anticancer agents and discuss the etiologies for the discrepancy between their preclinical and clinical evaluation. Finally, strategies for effective incorporation of MMPIs in current anticancer therapies are proposed.     

Marked defects in the expression and glycosylation of alpha2-HS glycoprotein/fetuin-A in plasma from neonates with intrauterine growth restriction: proteomics screening and potential clinical implications

Intrauterine growth restriction (IUGR) has been associated with increased perinatal morbidity and mortality and increased morbidity and metabolic abnormalities later in life. IUGR is characterized as the failure of a fetus to achieve his or her genetic growth potential in utero. Altered protein expression profiles associated with IUGR may be informative on the pathologic mechanisms of this condition and might reveal potential markers for postnatal complications. The aim of this study was to compare protein profiles of umbilical cord plasma from IUGR and appropriate for gestational age full-term neonates. Blood samples from doubly clamped umbilical cord at delivery from 10 IUGR and 10 appropriate for gestational age full-term neonates were analyzed by two-dimensional electrophoresis and MS. Prominent changes of the alpha2-HS glycoprotein/fetuin-A were observed in IUGR cases. Specifically we showed that these changes occur primarily at the level of post-translational modifications of the protein. Using a combination of mass spectrometry and classical biochemical assays, single and heavy chain forms of fetuin-A were found to lack the normally present O-linked sialic acids in IUGR neonates. Fetuin A is a glycoprotein that has been associated with promotion of in vitro cell replication, fetal growth and osteogenesis, and protection from Gram-negative bacterial endotoxins. Prominent defects in glycosylation/sialylation of fetuin-A revealed by our study might be responsible for impaired function of fetuin-A, leading to deficient fetal growth, especially osteogenesis, and/or to the development of complications frequently seen later in the lives of IUGR neonates.     

Delving into the complex picture of Ti(IV)-citrate speciation in aqueous media: Synthetic, structural, and electrochemical considerations in mononuclear Ti(IV) complexes containing variably deprotonated citrate ligands

The aqueous reaction of TiCl₄ with citric acid at pH ∼ 4 (KOH), led to the surprising isolation of a species assembly K₃[Ti(C₆H₆O₇)₂(C₆H₅O₇)] · K₄[Ti(C₆H₅O₇)₂(C₆H₆O₇)] · 10H₂O (1). The same system at pH ∼ 3 (neocuproine), led to the crystalline material (C₁₄H₁₃N₂)₂[Ti(C₆H₆O₇)₃] · 5H₂O (2), while at pH 5.0 (NaOH), afforded Na₃[Ti(C₆H₆O₇)₂(C₆H₅O₇)] · 9H₂O (3). Analytical, spectroscopic and structural characterization of 1, 2 and 3 revealed their distinct nature exemplified by mononuclear complexes bearing variably deprotonated citrates bound to Ti(IV). Solid-state ¹³C MAS NMR spectroscopy in concert with solution ¹³C and ¹H NMR on 3 provided ample evidence for the existence of bound citrates of distinct coordination mode to the metal ion. Cyclic voltammetry defined the electrochemical signature of complex 2, thereby projecting the physicochemical profile of the species formulated by the aforementioned properties. Comparison of cyclic voltammetric data on available discrete Ti(IV)-citrate species depicts the electrochemical profile and an E_1/2 value trend of the species in that binary system’s aqueous speciation, further substantiating the redox behavior of mononuclear Ti(IV)-citrate species in a pH-sensitive fashion. Collectively, the well-defined discrete species in 1-3 reflect and corroborate a synthetically challenging yet complex pH-specific picture of the aqueous Ti(IV) chemistry with the physiological citric acid, and shed light on the pH-dependent speciation in the binary Ti(IV)-citrate system.     

Transgenic tobacco plants overexpressing polyamine oxidase are not able to cope with oxidative burst generated by abiotic factors

The molecular and biochemical mechanism(s) of polyamine (PA) action remain largely unknown. Transgenic tobacco plants overexpressing polyamine oxidase (PAO) from Zea mays exhibited dramatically increased expression levels of Mpao and high 1,3-diaminopropane (Dap) content. All fractions of spermidine and spermine decreased significantly in the transgenic lines. Although Dap was concomitantly generated with H(2)O(2) by PAO, the latter was below the detection limits. To show the mode(s) of H(2)O(2) scavenging, the antioxidant machinery of the transgenics was examined. Specific isoforms of peroxidase, superoxide dismutase and catalase were induced in the transgenics but not in the wild-type (WT), along with increase in activities of additional enzymes contributing to redox homeostasis. One would expect that because the antioxidant machinery was activated, the transgenics would be able to cope with increased H(2)O(2) generated by abiotic stimuli. However, despite the enhanced antioxidant machinery, further increase in the intracellular reactive oxygen species (ROS) by exogenous H(2)O(2), or addition of methylviologen or menadione to transgenic leaf discs, resulted in oxidative stress as evidenced by the lower quantum yield of PSII, the higher ion leakage, lipid peroxidation and induction of programmed cell death (PCD). These detrimental effects of oxidative burst were as a result of the inability of transgenic cells to further respond as did the WT in which induction of antioxidant enzymes was evident soon following the treatments. Thus, although the higher levels of H(2)O(2) generated by overexpression of Mpao in the transgenics, with altered PA homeostasis, were successfully controlled by the concomitant activation of the antioxidant machinery, further increase in ROS was detrimental to cellular functions and induced the PCD syndrome.     

Embryoid bodies from mouse stem cells express oxytocin receptor, Oct-4 and DAZL

Oxytocin is a nine amino acid peptide involved in a wide spectrum of physiological functions; predominantly those concerning reproduction and differentiation are of interest. Oxytocin receptors are expressed at early developmental stages of mammals, suggesting that oxytocin might be involved in the determination of the germ stem cell line, at the very early stages of mammalian development. In this respect, the proximate aim of the present study was to confirm and further analyze the existence of oxytocin receptors at a very early level of cell commitment, that is, the determination of germ cells derived from embryoid bodies. To achieve our purpose we have cultured mouse embryonic stem cells under conditions inducing formation of embryoid bodies. In this work, ES cells were allowed to aggregate in a novel medium, “Stefanidis medium” from day 0 to day 14 until formed EBs. RNA was isolated from EBs and using RT-PCR we showed that EBs expressed Oct-4, OTR, OT, and DAZL. To demonstrate simultaneous expression immunocytochemistry was preformed, in which EBs showed strong immunoreactivity for both, OTR and DAZL molecular markers. We found that 35% of the cells displayed OTR, using flow cytometry. In addition, this novel medium showed to increase OTR mRNA. We propose, that at least in murine induced embryoid bodies there is simultaneous expression of oxytocin receptors and germ cell markers (DAZL) in many cells (expressing Oct-4). We thus conclude that, the oxytocin might indeed be a molecule playing a leading role in germ cell determination.     

Secretion and uptake of TAT-fusion proteins produced by engineered mammalian cells

Background

Intracellular signaling can be regulated by the exogenous addition of physiological protein inhibitors coupled to the TAT protein transduction domain. Thus far experiments have been performed with purified inhibitors added exogenously to cells in vitro or administered in vivo. Production of secretable TAT-fusion proteins by engineered mammalian cells, their uptake, and route of entry has not been thoroughly investigated. Such methodology, if established, could be useful for transplantation purposes.

Methods

Secretion of TAT-fusion proteins from transfected mammalian cells was achieved by means of a signal peptide. Cell uptake and subcellular localization of TAT-fusion proteins were determined by immunoblotting and confocal microscopy.

Results

Engineered TAT-fusion proteins were secreted with variable efficiency depending on the nature of the protein fused to the TAT peptide. Secreted proteins were able to transduce unmanipulated cells. Their mechanism of entry into cells partly involves lipid rafts and a portion of the internalised protein is directed to the Golgi.

Conclusions

Generation of secretable TAT-coupled inhibitors of signaling pathways, able to transduce other cells can be achieved.

General significance

These results provide key information that will assist in the design of TAT-inhibitors and engineered cells in order to regulate cell function within tissues.     

Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells

Trastuzumab (Herceptin), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab.