IL-2-activated CD8+CD44high cells express both adaptive and innate immune system receptors and demonstrate specificity for syngeneic tumor cells

CD8(+) T cells depend on the alphabeta TCR for Ag recognition and function. However, Ag-activated CD8(+) T cells can also express receptors of the innate immune system. In this study, we examined the expression of NK receptors on a population of CD8(+) T cells expressing high levels of CD44 (CD8(+)CD44(high) cells) from normal mice. These cells are distinct from conventional memory CD8(+) T cells and they proliferate and become activated in response to IL 2 via a CD48/CD2-dependent mechanism. Before activation, they express low or undetectable levels of NK receptors but upon activation with IL-2 they expressed significant levels of activating NK receptors including 2B4 and NKG2D. Interestingly, the IL-2-activated cells demonstrate a preference in the killing of syngeneic tumor cells. This killing of syngeneic tumor cells was greatly enhanced by the expression of the NKG2D ligand Rae-1 on the target cell. In contrast to conventional CD8(+) T cells, IL-2-activated CD8(+)CD44(high) cells express DAP12, an adaptor molecule that is normally expressed in activated NK cells. These observations indicate that activated CD8(+)CD44(high) cells express receptors of both the adaptive and innate immune system and may play a unique role in the surveillance of host cells that have been altered by infection or transformation.     

Deletion of N-terminal amino acids from human lecithin:cholesterol acyltransferase differentially affects enzyme activity toward alpha- and beta-substrate lipoproteins

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via alpha-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the beta-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure-function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1-5) of human LCAT. Western blot analysis showed that the mutant proteins (Delta 1-Delta 5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Delta 1 mutant) resulted in a dramatic loss of alpha-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full beta-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Delta 2 mutant) abolished alpha-LCAT activity and reduced beta-LCAT activity to 12% of wild type. Nevertheless, LCAT Delta 1 and Delta 2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the alpha-LCAT cofactor apoA-I (in Delta 1) and/or loss of enzyme activity (in Delta 1-Delta 5). Since the Delta 1 and Delta 2 mutants retain their ability to bind substrate, other factor(s), such as decreased access to the substrate binding pocket, may be responsible for the loss of enzyme activity.     

Whole bone marrow transplantation induces angiogenesis following acute ischemia

Several recent studies have shown that purified subsets of bone marrow (BM) cells can differentiate into endothelial, cardiac, and other cell types. During coronary artery bypass graft (CABG) surgery, sternal BM is routinely discarded. To determine if this BM can be used to induce angiogenesis and augment perfusion of the cardiac tissues after CABG, a simplified and more practical approach of using whole BM extract was tried to determine whether it would be adequate for the induction of BM-derived angiogenesis in experimental acute limb ischemia. BM was prepared from FVB/N-TgN(TIE2 lacZ)182 Sato (Tie2-lacZ) or B6.129S7-Gtrosa 26 (Rosa 26) mice that express beta-galactosidase (beta-gal) in endothelial cells and most adult tissues, respectively. Acute limb ischemia was induced in either C57BL6/J or FVB/N mice by double ligation of the left femoral artery just distal to the profunda femoral artery branch. Occlusion of the ligated artery was verified by angiography. The study group (n = 31) received an intramuscular injection of 50 micro l containing 1 x 10(6) BM cells, 5 mm proximal to the site of ligation. Experimental controls (n = 21) had an intramuscular injection of 50 micro l of saline. Angiogenesis in the mice was assessed by histological analysis. BM-derived beta-gal(+) cells were observed to aggregate in the vicinity of the ligated artery and not in the injected musculature BM-derived endothelial cells were incorporated within capillaries and small size blood vessels near the site of ligation. Generation of BM-derived blood vessels in experimental acute limb ischemia does not require purification of specific subset of cells. The elimination of cell purification will enhance the ease of using BM transplantation in generating blood vessels.     

Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d- galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 &mgr;m recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was used. We determined the precision and accuracy of this method for both a recessed and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins. Results were consistent with published values and independent of the working electrode. The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.      

Unrestrained Spindle Elongation during Recovery from Spindle Checkpoint Activation in cdc15-2 Cells Results in Mis-Segregation of Chromosomes

During normal metaphase in Saccharomyces cerevisiae, chromosomes are captured at the kinetochores by microtubules emanating from the spindle pole bodies at opposite poles of the dividing cell. The balance of forces between the cohesins holding the replicated chromosomes together and the pulling force from the microtubules at the kinetochores result in the biorientation of the sister chromatids before chromosome segregation. The absence of kinetochore–microtubule interactions or loss of cohesion between the sister chromatids triggers the spindle checkpoint which arrests cells in metaphase. We report here that an MEN mutant, cdc15-2, though competent in activating the spindle assembly checkpoint when exposed to Noc, mis-segregated chromosomes during recovery from spindle checkpoint activation. cdc15-2 cells arrested in Noc, although their Pds1p levels did not accumulate as well as in wild-type cells. Genetic analysis indicated that Pds1p levels are lower in a mad2Δ cdc15-2 and bub2Δ cdc15-2 double mutants compared with the single mutants. Chromosome mis-segregation in the mutant was due to premature spindle elongation in the presence of unattached chromosomes, likely through loss of proper control on spindle midzone protein Slk19p and kinesin protein, Cin8p. Our data indicate that a slower rate of transition through the cell division cycle can result in an inadequate level of Pds1p accumulation that can compromise recovery from spindle assembly checkpoint activation.     

The habenula prevents helpless behavior in larval zebrafish

Animals quickly learn to avoid predictable danger. However, if pre-exposed to a strong stressor, they do not display avoidance even if this causes continued contact with painful stimuli [1, 2]. In rodents, lesioning the habenula, an epithalamic structure that regulates the monoaminergic system, has been reported to reduce avoidance deficits caused by inescapable shock [3]. This is consistent with findings that inability to overcome a stressor is accompanied by an increase in serotonin levels [4]. However, other studies conclude that habenula lesions cause avoidance deficits [5, 6]. These contradictory results may be caused by lesions affecting unintended regions [6]. To clarify the role of the habenula, we used larval zebrafish, whose transparency and amenability to genetic manipulation enables more precise disruption of cells. We show that larval zebrafish learn to avoid a light that has been paired with a mild shock but fail to do so when pre-exposed to inescapable shock. Photobleaching of habenula afferents expressing the photosensitizer KillerRed causes a similar failure in avoidance. Expression of tetanus toxin in dorsal habenula neurons is sufficient to prevent avoidance. We suggest that this region may signal the ability to control a stressor, and that its disruption could contribute to anxiety disorders.     

Comparative gene expression profiling analysis of urothelial carcinoma of the renal pelvis and bladder

Background

Tumor therapy mainly attacks the metabolism to interfere the tumor's anabolism and signaling of proliferative second messengers. However, the metabolic demands of different cancers are very heterogeneous and depend on their origin of tissue, age, gender and other clinical parameters. We investigated tumor specific regulation in the metabolism of breast cancer.

Methods

For this, we mapped gene expression data from microarrays onto the corresponding enzymes and their metabolic reaction network. We used Haar Wavelet transforms on optimally arranged grid representations of metabolic pathways as a pattern recognition method to detect orchestrated regulation of neighboring enzymes in the network. Significant combined expression patterns were used to select metabolic pathways showing shifted regulation of the aggressive tumors.

Results

Besides up-regulation for energy production and nucleotide anabolism, we found an interesting cellular switch in the interplay of biosynthesis of steroids and bile acids. The biosynthesis of steroids was up-regulated for estrogen synthesis which is needed for proliferative signaling in breast cancer. In turn, the decomposition of steroid precursors was blocked by down-regulation of the bile acid pathway.

Conclusion

We applied an intelligent pattern recognition method for analyzing the regulation of metabolism and elucidated substantial regulation of human breast cancer at the interplay of cholesterol biosynthesis and bile acid metabolism pointing to specific breast cancer treatment.     

Crystal structures of blasticidin S deaminase (BSD): implications for dynamic properties of catalytic zinc

The set of blasticidin S (BS) and blasticidin S deaminase (BSD) is a widely used selectable marker for gene transfer experiments. BSD is a member of the cytidine deaminase (CDA) family; it is a zinc-dependent enzyme with three cysteines and one water molecule as zinc ligands. The crystal structures of BSD were determined in six states (i.e. native, substrate-bound, product-bound, cacodylate-bound, substrate-bound E56Q mutant, and R90K mutant). In the structures, the zinc position and coordination structures vary. The substrate-bound structure shows a large positional and geometrical shift of zinc with a double-headed electron density of the substrate that seems to be assigned to the amino and hydroxyl groups of the substrate and product, respectively. In this intermediate-like structure, the steric hindrance of the hydroxyl group pushes the zinc into the triangular plane consisting of three cysteines with a positional shift of approximately 0.6 A, and the fifth ligand water approaches the opposite direction of the substrate with a shift of 0.4 A. Accordingly, the zinc coordination is changed from tetrahedral to trigonal bipyramidal, and its coordination distance is extended between zinc and its intermediate. The shift of zinc and the recruited water is also observed in the structure of the inactivated E56Q mutant. This novel observation is different in two-cysteine cytidine deaminase Escherichia coli CDA and might be essential for the reaction mechanism in BSD, since it is useful for the easy release of the product by charge compensation and for the structural change of the substrate.     

Patterns of rapd markers and heavy metal concentrations in Perna viridis (L.), collected from metal-contaminated and uncontaminated coastal waters: are they correlated with each other?

Genetic variation due to heavy metal contamination has always been an interesting topic of study. Because of the numerous contaminants being found in coastal and intertidal waters, there is always much discussion and argument as to which contaminant(s) caused the variations in the genetic structures of biomonitors. This study used a Single Primer Amplification Reaction (SPAR) technique namely Random Amplified Polymorphic DNA (RAPD) to determine the genetic diversity of the populations of the green-lipped mussel Perna viridis collected from a metal-contaminated site at Kg. Pasir Puteh and those from four relatively' uncontaminated sites (reference sites). Heavy metal levels (Cd, Cu, Pb and Zn) were also measured in the soft tissues and byssus of the mussels from all the sites. Cluster analyses employing UPGMA done based on the RAPD makers grouped the populations into two major clusters; the Bagan Tiang, Pantai Lido, Pontian and Kg. Pasir Puteh populations were in one cluster, while the Sg. Belungkor population clustered by itself. This indicated that the genetic diversity based on bands resulting from the use of all four RAPD primers on P. viridis did not indicate its potential use as a biomarker of heavy metal pollution in coastal waters. However, based on a correlation analysis between a particular metal and a band resulting from a specific RAPD primer revealed some significant (P < 0.01) correlations between the primers and the heavy metal concentrations in the byssus and soft tissues. Thus, the correlation between a particular metal and the bands resulting from the use of a specific RAPD primer on P. viridis could be used as biomonitoring tool of heavy metal pollution.