Fractalkine/CX3CL1 production by human airway smooth muscle cells: induction by IFN-gamma and TNF-alpha and regulation by TGF-beta and corticosteroids

Chemokine synthesis by airway smooth muscle cells (ASMC) may be an important process underlying inflammatory cell recruitment in airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Fractalkine (FKN) is a recently described CX(3)C chemokine that has dual functions, serving as both a cell adhesion molecule and a chemoattractant for monocytes and T cells, expressing its unique receptor, CX(3)CR1. We investigated FKN expression by human ASMC in response to the proinflammatory cytokines IL-1beta, TNF-alpha, and IFN-gamma, the T helper 2-type cytokines IL-4, IL-10, and IL-13, and the fibrogenic cytokine transforming growth factor (TGF)-beta. Neither of these cytokines alone had any significant effect on ASMC FKN production. Combined stimulation with IFN-gamma and TNF-alpha induced FKN mRNA and protein expression in a time- and concentration-dependent manner. TGF-beta had a significant inhibitory effect on cytokine-induced FKN mRNA and protein expression. Dexamethasone (10(-8)-10(-6) M) significantly upregulated cytokine-induced FKN mRNA and protein expression. Finally, we used selective inhibitors of the mitogen-activated protein kinases c-Jun NH(2)-terminal kinase (JNK) (SP-610025), p38 (SB-203580), and extracellular signal-regulated kinase (PD-98095) to investigate their role in FKN production. SP-610025 (25 microM) and SB-203580 (20 microM), but not PD-98095, significantly attenuated cytokine-induced FKN protein synthesis. IFN-gamma- and TNF-alpha-induced JNK phosphorylation remained unaltered in the presence of TGF-beta but was inhibited by dexamethasone, indicating that JNK is not involved in TGF-beta- or dexamethasone-mediated regulation of FKN production. In summary, FKN production by human ASMC in vitro is regulated by inflammatory and anti-inflammatory factors.     

Effect of protein binding on ultrafast DNA dynamics: characterization of a DNA:APE1 complex

Synthetic oligonucleotides with a fluorescent coumarin group replacing a basepair have been used in recent time-resolved Stokes-shift experiments to measure DNA dynamics on the femtosecond to nanosecond timescales. Here, we show that the APE1 endonuclease cleaves such a modified oligonucleotide at the abasic site opposite the coumarin with only a fourfold reduction in rate. In addition, a noncatalytic mutant (D210N) binds tightly to the same oligonucleotide, albeit with an 85-fold reduction in binding constant relative to a native oligonucleotide containing a guanine opposite the abasic site. Thus, the modified oligonucleotide retains substantial biological activity and serves as a useful model of native DNA. In the complex of the coumarin-containing oligonucleotide and the noncatalytic APE1, the dye's absorption spectrum is shifted relative to its spectrum in either water or within the unbound oligonucleotide. Thus the dye occupies a site within the DNA:protein complex. This result is consistent with modeling, which shows that the complex accommodates coumarin at the site of the orphaned base with little distortion of the native structure. Stokes-shift measurements of the complex show surprisingly little change in the dynamics within the 40 ps-40 ns time range.     

Cancer and anticancer therapy-induced modifications on metabolism mediated by carnitine system

An efficient regulation of fuel metabolism in response to internal and environmental stimuli is a vital task that requires an intact carnitine system. The carnitine system, comprehensive of carnitine, its derivatives, and proteins involved in its transformation and transport, is indispensable for glucose and lipid metabolism in cells. Two major functions have been identified for the carnitine system: (1) to facilitate entry of long-chain fatty acids into mitochondria for their utilization in energy-generating processes; (2) to facilitate removal from mitochondria of short-chain and medium-chain fatty acids that accumulate as a result of normal and abnormal metabolism. In cancer patients, abnormalities of tumor tissue as well as nontumor tissue metabolism have been observed. Such abnormalities are supposed to contribute to deterioration of clinical status of patients, or might induce cancerogenesis by themselves. The carnitine system appears abnormally expressed both in tumor tissue, in such a way as to greatly reduce fatty acid beta-oxidation, and in nontumor tissue. In this view, the study of the carnitine system represents a tool to understand the molecular basis underlying the metabolism in normal and cancer cells. Some important anticancer drugs contribute to dysfunction of the carnitine system in nontumor tissues, which is reversed by carnitine treatment, without affecting anticancer therapeutic efficacy. In conclusion, a more complex approach to mechanisms that underlie tumor growth, which takes into account the altered metabolic pathways in cancer disease, could represent a challenge for the future of cancer research.     

Engineering two mutants of cDNA-encoding G2 subunit of soybean glycinin capable of self-assembly <Emphasis Type="Italic">in vitro</Emphasis> and rich in methionine

The main goal of this work was to make the cDNA-encoding subunit G2 of soybean glycinin, capable of self-assembly in vitro and rich in methionine residues. Two mutants (pSP65/G4SacG2 and pSP65/G4SacG2HG4) were therefore constructed. The constructed mutants were successfully assembled in vitro into oligomers similar to those occurred in the seed. The successful self-assembly was due to the introduction of Sac fragment of Gy4 (the codons of the first 21 amino acid residues), which reported to be the key element in self-assembly into trimers. The mutant pSP65/G4SacG2HG4 included the acidic chain of Gy4 (HG4), which was previously molecularly modified to have three methionine residues. This mutant will be useful in the efforts to improve the seed quality.     

Statistically quantified measurement of an Alzheimer's marker by surface‐enhanced Raman scattering

Fibrillar forms of the Amyloid‐β (Aβ) protein have been implicated in the early stages of Alzheimer's disease (AD), however there are no standardised assays for soluble Aβ oligomer biomarkers that provide the best indication of the disease progression [1,2]. As a step towards a fast and label‐free method for testing different AD biomarkers, we have combined laser nano‐textured substrates with a SERS mapping technique and validated it using soluble Aβ‐40 oligomers [3‐5]. The nano‐textured SERS substrates provide fast (&5 min), label‐free spectra associated with soluble Aβ‐40 oligomers down to a concentration of 10 nM. Statistical analysis of the spectral intensities mapped over the substrate surface shows a quantitative correlation with the oligomer concentration.

Schematics of experiments: SERS mapping of Aβ‐40 (left figure: measured SERS intensity overlayed with an SEM image of ripples) was carried out on the laser nano‐textured (ripple) surface of sapphire and statistical analysis of the SERS intensity was carried out for qualitative (a high SERS intensity at low probability) and quantitative (a moderate SERS intenisty at the highest probability) measures. Quantitative statistical analysis of SERS mapping data can be performed off line for cross correlations with other known SERS signatures.