An aminomethylpyrimidine DPP-IV inhibitor with improved properties

A recently identified DPP-IV inhibitor (1) was found to induce phospholipidosis and to inhibit CYP3A4. A small series of less lipophilic and less amphiphilic analogues was synthesized in an effort to overcome these issues. One compound from this series was equipotent to 1, did not induce phospholipidosis and showed a reduced CYP3A4 inhibition.     

E1A stimulates FGF-2 release promoting differentiation of primary endothelial cells

Basic Fibroblast Growth Factor (FGF-2) is a growth and survival factor and represents one of the most potent differentiation agents of vascular system. In the present study we describe that adenoviral oncoprotein E1A regulates FGF-2 production and determines the acquisition of a pro-angiogenic phenotype in primary bovine aortic endothelial cells (BAEC). Following their transfection, wild type E1A proteins 12S and 13S (wtE1A) stimulated BAEC to differentiate on reconstituted basement membrane matrix (Matrigel). This outcome was paralleled by invasion and migration enhancement in wtE1A-transfected cells. This stimulating effect was absent with the E1A mutant dl646N. Accordingly, zymography and RT - PCR analyses showed that matrix metalloproteinase-9 protein- and mRNA-levels increased following wtE1A transfection. Interestingly, wtE1A-transfected BAEC showed FGF-2 mRNA- and protein-levels higher than controls. Further, FGF-2 neutralization reduced the amount of MMP-9 released in the supernatant of E1A-transfected cells and strongly inhibited BAEC differentiation, thus suggesting that wtE1A activates BAEC by a mechanism, at least partially, dependent on a FGF-2 autocrine/paracrine loop.     

Evidence for non-enzymatic glycosylation of Escherichia coli chromosomal DNA

We have recently shown that the process of non-enzymatic glycosylation (glycation) takes place in Escherichia coli under physiological conditions and affects both recombinant and endogenous bacterial proteins. In this study, we further demonstrate that E. coli chromosomal DNA is also subjected to glycation under physiological growth conditions. The E. coli DNA accumulates early glycation (Amadori) products as proven by the nitroblue tetrazolium (NBT) reduction assay. It showed also immunoreactivity to a monoclonal antibody raised against N(in)-(carboxymethyl)lysine and fluorescent properties indicative of modifications with advanced glycation end-products. Two types of fluorophores were detected in the E. coli DNA with excitation maxima at 360 nm and 380 nm and emission maxima at 440 nm and 410 nm. Using the NBT reduction assay, fluorescence spectroscopy and enzyme-linked immunosorbent assay we revealed that glycation adducts accumulate in DNA predominantly in the stationary phase of growth, although they could be detected also in exponential-phase cells. Besides on the growth phase, the extent of DNA glycation depends also on the nutrient broth composition being more extensive in rich media. Thiamine was found to inhibit both DNA glycation and spontaneous point mutations as judged by the decreased rate of the argE3 to Arg(+) reversions in the E. coli strain AB1157.     

The Toxoplasma gondii bradyzoite antigens BAG1 and MAG1 induce early humoral and cell-mediated immune responses upon human infection

Infection of humans by Toxoplasma gondii leads to an acute systemic phase, in which tachyzoites disseminate throughout the body, followed by a chronic phase characterized by the presence of tissue cysts, containing bradyzoites, in brain, heart and skeletal muscles. This work focused on studying the antigenic regions of bradyzoite-specific proteins involved in human B- and T-cell responses. To this aim, we constructed a phage-display library of DNA fragments derived from the bradyzoite-specific genes BAG1, MAG1, SAG2D, SAG4, BSR4, LDH2, ENO1 and p-ATPase. Challenge of the bradyzoite library with sera of infected individuals led to the identification of antigenic regions within BAG1 and MAG1 gene products. Analysis of the humoral and lymphoproliferative responses to recombinant antigens demonstrated that the BAG1 fragment induced T-cell proliferation in 34% of T. gondii-exposed individuals, while 50% of them had specific IgG. In the same subjects, the MAG1 fragment was recognized by T cells from 17% of the exposed donors and by antibodies from 73% of them. A detailed analysis of the antibody response against BAG1 and MAG1 antigen fragments demonstrated that the immune response against bradyzoites occurs early after infection in humans. Finally, we provide evidence that the T-cell response against BAG1 is associated with the production of interferon-gamma, suggesting that bradyzoite antigens should be considered in the design of potential vaccines in humans.     

Establishment of a cell model of ALS disease: Golgi apparatus disruption occurs independently from apoptosis

The Golgi apparatus (GA) appears disrupted in motor neurons of amyotrophic lateral sclerosis (ALS). Here, mouse motor neuron-like NSC-34 cell lines stably expressing human superoxide dismutase 1 (hSOD1)^wt and mutant hSOD1^G93A, as an ALS cell model, were constructed. The number of cells with disrupted GA increased from 14% to 34%. Furthermore, NSC-34/hSOD1^G93A cells showed lower levels of proliferation and differentiation. GA disruption was not caused by apoptosis as determined by several techniques including caspase-3 activation. Similarly, spinal cords from ALS patients did not show caspase-3 activation. Therefore, NSC-34/hSOD1^G93A cells are a suitable cell model to study GA dysfunction in ALS.     

Involvement of carbohydrates in the hardening of the zona pellucida of mouse oocytes

The effect of lectins with different saccharide specificity (ConA, LCA, DBA, WGA and PNA) on enzymatic digestion of the zona pellucida (ZP) of mouse oocytes was studied. All lectins tested, except PNA, induced ZP hardening with different degrees of efficiency. Moreover, extensive ZP digestion with mixed exoglycosidase prevented "spontaneous" ZP hardening. These observations suggest that changes of the carbohydrate moieties can be involved in the hardening of the zona pellucida of mouse oocytes.