Binding partners for the COOH-terminal appendage domains of the GGAs and gamma-adaptin

The adaptor appendage domains are believed to act as binding platforms for coated vesicle accessory proteins. Using glutathione S-transferase pulldowns from pig brain cytosol, we find three proteins that can bind to the appendage domains of both the AP-1 gamma subunit and the GGAs: gamma-synergin and two novel proteins, p56 and p200. p56 elicited better antibodies than p200 and was generally more tractable. Although p56 and gamma-synergin bind to both GGA and gamma appendages in vitro, immunofluorescence labeling of nocodazole-treated cells shows that p56 colocalizes with GGAs on TGN46-positive membranes, whereas gamma-synergin colocalizes with AP-1 primarily on a different membrane compartment. Furthermore, in AP-1-deficient cells, p56 remains membrane-associated whereas gamma-synergin becomes cytosolic. Thus, p56 and gamma-synergin show very strong preferences for GGAs and AP-1, respectively, in vivo. However, the GGA and gamma appendages share the same fold as determined by x-ray crystallography, and mutagenesis reveals that the same amino acids contribute to their binding sites. By overexpressing wild-type GGA and gamma appendage domains in cells, we can drive p56 and gamma-synergin, respectively, into the cytosol, suggesting a possible mechanism for selectively disrupting the two pathways.     

The Arabidopsis Cdc2a-interacting protein ICK2 is structurally related to ICK1 and is a potent inhibitor of cyclin-dependent kinase activity in vitro

Cyclin-dependent kinases (CDKs) are important regulators of the eukaryotic cell division cycle. To study protein-protein interactions involving plant CDKs, the Arabidopsis thaliana Cdc2aAt was used as bait in the yeast two-hybrid system. Here we report on the isolation of ICK2, and show that it interacts with Cdc2aAt, but not with a second CDK from Arabidopsis, Cdc2bAt. ICK2 contains a carboxy-terminal domain related to that of ICK1, a previously described CDK inhibitor from Arabidopsis, and to the CDK-binding domain of the mammalian inhibitor p27Kip1. Outside of this domain, ICK2 is distinct from ICK1, p27Kip1, and other proteins. At nanogram levels (8 nM), purified recombinant ICK2 inhibits p13Suc1-associated histone H1 kinase activity from Arabidopsis tissue extracts, demonstrating that it is a potent inhibitor of plant CDK activity in vitro. ICK2 mRNA was present in all tissues analysed by Northern hybridization, and its distribution was distinct from that of ICK1. These results demonstrate that plants possess a family of differentially regulated CDK inhibitors that contain a conserved carboxy terminal but with distinct amino terminal regions.     

The clinical significance of thymidine kinase 1 measurement in serum of breast cancer patients using anti-TK1 antibody

The activity of total thymidine kinase in serum (S-TK) has been used as a tumor maker for decades. To date such activity has been determined using [125]I-iodo-deoxyuridine as a substrate. The aim of this study was to develop a new, antibody-based technique for the measurement of cytoplasmic thymidine kinase (TK1) in serum. Both mono- and polyclonal antibodies against S-TK1 were used in dot blot assay. S-TK1 was characterized by SDS and IEF techniques. Sixty-five breast cancer patients were studied, including 17 preoperative and 38 postoperative tumor-free patients and 10 patients with metastases to the lymph nodes (N1-2). They were compared to patients with benign tumors (n=21) and healthy volunteers (n=11). S-TK1 was low (0-1.0 pM) in healthy volunteers, while in preoperative patients the level was increased 6-110-fold. Significant differences were observed between preoperative patients and healthy volunteers (p=0.005), preoperative patients and patients with benign tumors (p<0.001), and preoperative patients and postoperative patients without metastases (p<0.001). No significant difference was observed between preoperative patients and postoperative patients with metastases (p=0.191). The S-TK activity in preoperative patients was also high in serum, but no decrease was observed following surgery. In conclusion, the anti-TK1 antibody could be a good marker for monitoring the response of breast cancer patients to therapy.     

Further characterization of the binding of human recombinant interleukin 2 to heparin and identification of putative binding sites

We have previously provided compelling evidence that human recombinant interleukin 2 (IL-2) binds to the sulfated polysaccharides heparin, highly sulfated heparan sulfate and fucoidan. Here we show that IL-2 binding is dependent on heparin chain length, but with fragments as small as 15-mers retaining binding activity. The addition of exogenous heparin has no effect on the in vitro biological activity of IL-2. In addition soluble IL-2 receptor alpha and beta polypeptides do not compete with heparin for the binding of IL-2. IL-2 bound by heparin is still recognized by two IL-2 specific monoclonal antibodies, 3H9 and H2- 8, whose epitopes lie in the amino terminal region. Murine IL-2 unlike its human counterpart fails to bind to heparin. Human IL-2 analogs with single amino acid substitutions at positions Lys43, Thr51, and Gln126 analogs no longer bind to heparin. By contrast the Arg38Ala analog retains heparin full heparin binding activity. These experimental findings together with molecular modeling studies suggest two putative heparin binding sites on human IL-2, one involving four basic residues, Lys48, Lys49, Lys54, and His55, and the other being a discontinuous site comprising Lys43, Lys64, Arg81, and Arg83. Neither of these two clusters is completely conserved in murine IL-2. Overall our data suggest that the binding of human IL-2 to heparin and heparan sulfate does not interfere with IL-2/IL-2 receptor interactions. Therefore, binding to glycosaminoglycan may be a mechanism for retaining the cytokine in an active form close to its site of secretion in the tissue, thus favoring a paracrine role for IL-2.      

Post-Operative Plasma Osteopontin Predicts Distant Metastasis in Human Colorectal Cancer

BackgroundThe overall prognosis of colorectal cancer (CRC) patients is unsatisfactory due to cancer metastasis after operation. This study aims to investigate the clinical significance of plasma osteopontin (OPN) levels as minimally invasive, predictive, and surrogate biomarkers for prognosis of CRC patients.MethodsThis randomized study design consists of pre-operative and post-operative plasma samples from a total of 79 patients. We determined plasma levels of OPN by ELISA and examined their correlation with the clinicopathological parameters of CRC patients. The effects of endogenous and exogenous OPN on CRC metastasis were investigated by examination of the effect on regulators of epithelial to messenchymal transition and migration assay.ResultsOur findings demonstrated for the first time the clinical correlation of plasma OPN with metastasis of CRC patients. High post-operative plasma OPN level (>153.02 ng/ml) associated with development of metastasis after curative resection (p<0.001). Moreover, post-operative plasma OPN level correlated with disease-free survival of CRC patients (p=0.009) and was an independent factor for predicting development of metastasis in CRC patients after curative resection (p=0.036). Our in vitro model showed that OPN ectopic expression induced DLD1 cell migration through Snail and Twist1 overexpression and E-cadherin repression, and secretory OPN level enhanced cell migration.ConclusionsThe results of the current study suggest that post-operative plasma OPN correlated with post-operative metastasis, suggesting that it is a potential non-invasive biomarker for the development of future metastasis in CRC patients. In addition, OPN was shown to be involved in the metastatic process and thus inhibition of OPN is a potential therapeutic approach to treat CRC patients.     

Traveling wave solutions from microscopic to macroscopic chemotaxis models

In this paper, we study the existence and nonexistence of traveling wave solutions for the one-dimensional microscopic and macroscopic chemotaxis models. The microscopic model is based on the velocity jump process of Othmer et al. (SIAM J Appl Math 57:1044–1081, 1997). The macroscopic model, which can be shown to be the parabolic limit of the microscopic model, is the classical Keller–Segel model, (Keller and Segel in J Theor Biol 30:225–234; 377–380, 1971). In both models, the chemosensitivity function is given by the derivative of a potential function, Φ(v), which must be unbounded below at some point for the existence of traveling wave solutions. Thus, we consider two examples: F(v) = lnv{\Phi(v) = \ln v} and F(v) = ln[v/(1-v)]{\Phi(v) = \ln[v/(1-v)]}. The mathematical problem reduces to proving the existence or nonexistence of solutions to a nonlinear boundary value problem with variable coefficient on \mathbb R{\mathbb R}. The main purpose of this paper is to identify the relationships between the two models through their traveling waves, from which we can observe how information are lost, retained, or created during the transition from the microscopic model to the macroscopic model. Moreover, the underlying biological implications of our results are discussed.     

Efflux pumps expression and its association with porin down-regulation and β-lactamase production among <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> causing bloodstream infections in Brazil

Background  

Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile.     

Multi-parametric neuroimaging evaluation of cerebrotendinous xanthomatosis and its correlation with neuropsychological presentations

Background

Cerebrotendinous xanthomatosis (CTX) is a rare genetic disorder. Recent studies show that brain damage in CTX patients extends beyond the abnormalities observed on conventional magnetic resonance imaging (MRI). We studied the MRI and ^{99 m}Tc-ethyl cysteinate dimer single photon emission computed tomography (SPECT) findings of CTX patients and made a correlation with the neuropsychological presentations.

Methods

Diffusion tensor imaging (DTI) and 3D T1-weighted images of five CTX patients were compared with 15 age-matched controls. Voxel-based morphometry (VBM) was use to delineate gray matter (GM) and white matter (WM) volume loss. Fractional anisotropy (FA), mean diffusivity (MD), and eigenvalues derived from DTI were used to detect WM changes and correlate with neuropsychological results. SPECT functional studies were used to correlate with GM changes.

Results

Cognitive results showed that aside from moderate mental retardation, the patient group performed worse in all cognitive domains. Despite the extensive GM atrophy pattern, the cerebellum, peri-Sylvian regions and parietal-occipital regions were correlated with SPECT results. WM atrophy located in the peri-dentate and left cerebral peduncle areas corresponded with changes in diffusion measures, while axial and radial diffusivity suggested both demyelinating and axonal changes. Changes in FA and MD were preceded by VBM in the corpus callosum and corona radiata. Cognitive results correlated with FA changes.

Conclusion

In CTX, GM atrophy affected the perfusion patterns. Changes in WM included atrophy, and axonal changes with demyelination. Disconnection of major fiber tracts among different cortical regions may contribute to cognitive impairment.

     

Inclusion complexes of pyrimethamine in 2-hydroxypropyl-beta-cyclodextrin: characterization, phase solubility and molecular modelling

The inclusion complexation of pyrimethamine in 2-hydroxypropyl-beta-cyclodextrin has been investigated by 2D (1)H NMR, FTIR and UV/visible spectroscopy and also by molecular modelling methods (AM1, PM3, MM3). From the phase-solubility diagram a linear increase was observed in pyrimethamine aqueous solubility in the presence of 2-hydroxypropyl-beta-cyclodextrin, evidencing the formation of a soluble inclusion complex. According to the continuous variation method (Job's plot) applied to fluorescence measurements, a 1:1 stoichiometry has been proposed for the complex. Concerning the structure of the complex, a Cl-in orientation of pyrimethamine in the 2-hydroxypropyl-beta-cyclodextrin cavity has been proposed from the theoretical calculations, being confirmed by two-dimensional (1)H NMR spectroscopy (ROESY). The thermal behaviour has also been studied, providing complementary evidences of complex formation.     

Antiadhesive property of microalgal polysaccharide extract on the binding of Helicobacter pylori to gastric mucin

The emergence of antibiotic-resistant Helicobacter pylori is of concern in the treatment of H. pylori-associated gastroduodenal diseases. As the organism was reported to bind gastric mucin, we used porcine gastric mucin as substrate to assess the antiadhesive property of polysaccharides derived from Spirulina (PS), a commercially available microalga, against the binding of H. pylori to gastric mucin. Results show that polysaccharides prevented H. pylori from binding to gastric mucin optimally at pH 2.0, without affecting the viability of either bacteria or gastric epithelial cells, thus favouring its antiadhesive action in a gastric environment. Using ligand overlay analysis, polysaccharide was demonstrated to bind H. pylori alkyl hydroperoxide reductase (AhpC) and urease, which have shown here to possess mucin-binding activity. An in vivo study demonstrated that bacteria load was reduced by >90% in BALB/c mice treated with either Spirulina or polysaccharides. It is thus suggested that polysaccharides may function as a potential antiadhesive agent against H. pylori colonization of gastric mucin.