The tandem chain extension aldol reaction used for synthesis of ketomethylene tripeptidomimetics targeting hPEPT1

The rationale for targeting the human di-/tripeptide transporter hPEPT1 for oral drug delivery has been well established by several drug and prodrug cases. The aim of this study was to synthesize novel ketomethylene modified tripeptidomimetics and to investigate their binding affinity for hPEPT1. Three related tripeptidomimetics of the structure H-Phe-ψ[COCH₂]-Ser(Bz)-X_aa-OH were synthesized applying the tandem chain extension aldol reaction, where amino acid derived β-keto imides were stereoselectively converted to α-substituted γ-keto imides. In addition, three corresponding tripeptides, composed of amide bonds, were synthesized for comparison of binding affinities. The six investigated compounds were all defined as high affinity ligands (K_i-values <0.5 mM) for hPEPT1 by measuring the concentration dependent inhibition of apical [¹⁴C]Gly-Sar uptake in Caco-2 cells. Consequently, the ketomethylene replacement for the natural amide bond and α-side chain modifications appears to offer a promising strategy to modify tripeptidic structures while maintaining a high affinity for hPEPT1.     

Adipose tissue fatty acid patterns and changes in anthropometry: a cohort study

Introduction

Diets rich in n-3 long chain polyunsaturated fatty acids (LC-PUFA), but low in n-6 LC-PUFA and 18:1 trans-fatty acids (TFA), may lower the risk of overweight and obesity. These fatty acids have often been investigated individually. We explored associations between global patterns in adipose tissue fatty acids and changes in anthropometry.

Methods

34 fatty acid species from adipose tissue biopsies were determined in a random sample of 1100 men and women from a Danish cohort study. We used sex-specific principal component analysis and multiple linear regression to investigate the associations of adipose tissue fatty acid patterns with changes in weight, waist circumference (WC), and WC controlled for changes in body mass index (WC_BMI), adjusting for confounders.

Results

7 principal components were extracted for each sex, explaining 77.6% and 78.3% of fatty acid variation in men and women, respectively. Fatty acid patterns with high levels of TFA tended to be positively associated with changes in weight and WC for both sexes. Patterns with high levels of n-6 LC-PUFA tended to be negatively associated with changes in weight and WC in men, and positively associated in women. Associations with patterns with high levels of n-3 LC-PUFA were dependent on the context of the rest of the fatty acid pattern.

Conclusions

Adipose tissue fatty acid patterns with high levels of TFA may be linked to weight gain, but patterns with high n-3 LC-PUFA did not appear to be linked to weight loss. Associations depended on characteristics of the rest of the pattern.     

Detection of nucleotide binding to Na,K-ATPase in an aqueous membrane suspension by 13C cross-polarization magic-angle spinning NMR spectroscopy

Binding of uniformly (13)C labelled ATP to Na,K-ATPase was studied by (13)C cross-polarization magic-angle spinning (CP-MAS) NMR. In the presence of 30 mM Na(+) , and with sample- and time-averaging, NMR spectra obtained at 4 degrees C exhibited several resonances for the bound nucleotide. Chemical shifts suggested that site-specific changes in the micro-environment or conformation of the nucleotide occurred in the high affinity binding site. These experiments permit further studies of nucleotide dynamics, structure and binding under physiologically relevant conditions.     

Quantifying and imaging NY-ESO-1/LAGE-1-derived epitopes on tumor cells using high affinity T cell receptors

Presentation of intracellular tumor-associated Ags (TAAs) in the context of HLA class I molecules offers unique cancer-specific cell surface markers for the identification and targeting of tumor cells. For most peptide Ags, the levels of and variations in cell surface presentation remain unknown, yet these parameters are of crucial importance when considering specific TAAs as targets for anticancer therapy. Here we use a soluble TCR with picomolar affinity for the HLA-A2-restricted 157-165 epitope of the NY-ESO-1 and LAGE-1 TAAs to investigate presentation of this immunodominant epitope on the surface of a variety of cancer cells. By single molecule fluorescence microscopy, we directly visualize HLA-peptide presentation for the first time, demonstrating that NY-ESO-1/LAGE-1-positive tumor cells present 10-50 NY-ESO-1/LAGE-1(157-165) epitopes per cell.     

Stem cells from the adult human brain develop into functional neurons in culture

Recent research communications indicate that the adult human brain contains undifferentiated, multipotent precursors or neural stem cells. It is not known, however, whether these cells can develop into fully functional neurons. We cultured cells from the adult human ventricular wall as neurospheres and passed them at the individual cell level to secondary neurospheres. Following dissociation and plating, the cells developed the antigen profile of the three main cell types in the brain (GFAP, astrocytes; O2, oligodendrocytes; and beta-III-tubulin/NeuN, neurons). More importantly, the cells developed the electrophysiological profiles of neurons and glia. Over a period of 3 weeks, neuron-like cells went through the same phases as neurons do during development in vivo, including up-regulation of inward Na+ -currents, drop in input resistance, shortening of the action potential, and hyperpolarization of the cell membrane. The cells developed overshooting action potentials with a mature configuration. Recordings in voltage-clamp mode displayed both the fast inactivating TTX-sensitive sodium current (INa) underlying the rising phase of the action potential and the two potassium currents terminating the action potential in mature neurons (IA and IK, sensitive to 4-AP and TEA, respectively). We have thus demonstrated that the human ventricular wall contains multipotent cells that can differentiate into functionally mature neurons.     

Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene

The ability of herpes simplex virus type 1 thymidine kinase (HSV-tk)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-tk-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs via the transfer of phosphorylated GCV, the mechanism(s) of this bystander effect and the importance of gap junctions for the effect of prodrug/suicide gene therapy in primary human glioblastoma cells remains elusive. Surgical biopsies of malignant gliomas were used to establish explant primary cultures. Proliferating tumor cells were characterized immunohistochemically and found to express glial tumor markers including nestin, vimentin, glial fibrillary acidic protein (GFAP), S-100, and gap junction protein connexin 43 (Cx43). Western blot analysis revealed the presence of phosphorylated isoforms of Cx43 and Calcein/DiI fluorescent dye transfer showed evidence of efficient gap junction communication (GJC). In order to study the effect(s) of prodrug/suicide gene therapy in these cultures, human glioblastoma cell cultures were transfected with the HSVtk gene for transient or stable expression. Ganciclovir treatment of these cultures led to >90% of cells dead within 1 week. Eradication of cells could be inhibited by the addition of alpha-glycyrrhetinic acid (AGA), a GJC inhibitor. In parallel experiments, AGA decreased the immunodetection of phosphorylated Cx43 as analyzed by Western blot and inhibited fluorescent dye transfer. In conclusion, these observations are consistent with GJC as the mediator of the bystander effect in primary cultures of human glioblastoma cells by the transfer of phosphorylated GCV from HSVtk gene transfected cells to untransfected ones.     

Nucleic acid capture assay,a new method for direct quantitation of nucleic acids

Technologies allowing direct detection of specific RNA/DNA sequences occasionally serve as an alternative to amplification methods for gene expression studies. In these direct methods the hybridization of probes takes place in complex mixtures, thus specificity and sensitivity still limit the use of current technologies. To address these challenges, we developed a new technique called the nucleic acid capture assay, involving a direct multi-capture system. This approach combines a 3′-ethylene glycol scaffolding with the incorporation of 2′-methoxy deoxyribonucleotides in the capture sequences. In our design, all nucleotides other than those complementary to the target mRNA have been replaced by an inert linker, resulting in significant reductions in non-specific binding. We also provide a versatile method to detect the presence of captured targets by using specific labeled probes with alkaline phosphatase-conjugated anti-label antibodies. This direct, flexible and reliable technique for gene expression analysis is well suited for high-throughput screening and has potential for DNA microarray applications.     

Caspase-mediated parkin cleavage in apoptotic cell death

The parkin protein is important for the survival of the neurons that degenerate in Parkinson's disease as demonstrated by disease-causing lesions in the parkin gene. The Chinese hamster ovary and the SH-SY5Y cell line stably expressing recombinant human parkin combined with epitope-specific parkin antibodies were used to investigate the proteolytic processing of human parkin during apoptosis by immunoblotting. Parkin is cleaved during apoptosis induced by okadaic acid, staurosporine, and camptothecin, thereby generating a 38-kDa C-terminal fragment and a 12-kDa N-terminal fragment. The cleavage was not significantly affected by the disease-causing mutations K161N, G328E, T415N, and G430D and the polymorphism R366W. Parkin and its 38-kDa proteolytic fragment is preferentially associated with vesicles, thereby indicating that cleavage is a membrane-associated event. The proteolysis is sensitive to inhibitors of caspases. The cleavage site was mapped by site-directed mutagenesis of potential aspartic residues and revealed that mutation of Asp-126 alone abrogated the parkin cleavage. The tetrapeptide aldehyde LHTD-CHO, representing the amino acid sequence N-terminal to the putative cleavage site was an efficient inhibitor of parkin cleavage. This suggests that parkin function is compromised in neuropathological states associated with an increased caspase activation, thereby further adding to the cellular stress.     

Functional diversity of arbuscular mycorrhizas extends to the expression of plant genes involved in P nutrition

This study of functional diversity considers symbiotic associations between two plant species, Medicago truncatula and Lycopersicon esculentum, and seven species of arbuscular mycorrhizal fungi (AMF). The objective was to integrate physiological analyses with molecular techniques to test whether functional diversity between AMF species is not only apparent at the level of mycorrhiza formation, plant nutrient uptake and plant growth, but also at the molecular level as observed by variation in the root expression of plant genes involved in the plant's P-starvation response. The seven species of AMF varied widely in their influence on the root expression of MtPT2 and Mt4 from M. truncatula and LePT1 and TPSI1 from L. esculentum. At one extreme was Glomus mosseae, whereby its colonization of M. truncatula resulted in the greatest reduction in MtPT2 and Mt4 gene expression and the highest level of P uptake and growth, while at the other extreme was Gigaspora rosea, whereby colonization resulted in the highest levels of MtPT2 and Mt4 gene expression and the lowest P uptake and growth. The expression of LePT1 and TPSI1 within the roots of L. esculentum was low and relatively uniform across the seven mycorrhizas, reflecting the ability of this cultivar to maintain low and constant shoot P levels despite root colonization by a broad selection of AMF. This study extends current understanding of functional diversity and shows that plants can respond differently to AMF, not only at the level of colonization, nutrient uptake and growth, but also at the level of gene expression.